simple_bioc 0.0.11 → 0.0.12

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  1. checksums.yaml +4 -4
  2. data/lib/simple_bioc/bioc_merger.rb +2 -37
  3. data/lib/simple_bioc/bioc_reader.rb +2 -0
  4. data/lib/simple_bioc/location_adjuster.rb +39 -0
  5. data/lib/simple_bioc/passage.rb +2 -0
  6. data/lib/simple_bioc/sentence.rb +2 -0
  7. data/lib/simple_bioc/version.rb +1 -1
  8. data/spec/file_check_spec.rb +10 -2
  9. data/xml/10330397_gene.xml +1 -0
  10. data/xml/10330397_ppimention.xml +1442 -0
  11. data/xml/merge/10366597_error.xml +3 -0
  12. data/xml/merge/output_10330397.xml +6447 -0
  13. data/xml/merge/output_10366597.xml +1360 -0
  14. metadata +8 -2
data/xml/10330397_gene.xml ADDED
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+ <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE collection SYSTEM "BioC.dtd"><collection><source>PMC</source><date>20140719</date><key>ascii.key</key><document><id>2133178</id><passage><infon key="journal-title">The Journal of Cell Biology</infon><infon key="name_3">surname:Gimeno;given-names:Ruth</infon><infon key="kwd">endoplasmic reticulum transport vesicle COPII proton-ATPase Saccharomyces cerevisiae</infon><infon key="name_4">surname:Kaiser;given-names:Chris A.</infon><infon key="name_2">surname:Espenshade;given-names:Peter</infon><infon key="article-id_pmid">10330397</infon><infon key="name_1">surname:Crotwell;given-names:Michelle</infon><infon key="name_0">surname:Roberg;given-names:Kevin J.</infon><infon key="issue">4</infon><infon key="volume">145</infon><infon key="article-id_pmc">2133178</infon><infon key="year">1999</infon><infon key="type">front</infon><infon key="fpage">659</infon><infon key="lpage">672</infon><offset>0</offset><sentence><offset>0</offset><text>LST1 Is a SEC24 Homologue Used for Selective Export of the Plasma Membrane ATPase from the Endoplasmic Reticulum</text><annotation id="Team286_A1"><infon key="type">Gene</infon><location offset="0" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A2"><infon key="type">Gene</infon><location offset="10" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A3"><infon key="type">Gene</infon><location offset="59" length="22"></location><text>Plasma Membrane ATPase</text></annotation></sentence></passage><passage><infon key="type">abstract</infon><offset>114</offset><sentence><offset>114</offset><text>In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins.</text><annotation id="Team286_A4"><infon key="type">Gene</infon><location offset="231" length="19"></location><text>COPII coat proteins</text></annotation></sentence><sentence><offset>252</offset><text>We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1.</text><annotation id="Team286_A5"><infon key="type">Gene</infon><location offset="302" length="3"></location><text>LST</text></annotation><annotation id="Team286_A6"><infon key="type">Gene</infon><location offset="307" length="11"></location><text>lethal with</text></annotation><annotation id="Team286_A7"><infon key="type">Gene</infon><location offset="375" length="23"></location><text>COPII mutation sec13-1</text></annotation></sentence><sentence><offset>400</offset><text>LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function.</text><annotation id="Team286_A8"><infon key="type">Gene</infon><location offset="400" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A9"><infon key="type">Gene</infon><location offset="467" length="11"></location><text>COPII genes</text></annotation><annotation id="Team286_A10"><infon key="type">Gene</infon><location offset="497" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>532</offset><text>LST1 codes for a protein similar in sequence to the COPII subunit Sec24p.</text><annotation id="Team286_A11"><infon key="type">Gene</infon><location offset="532" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>608</offset><text>Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p.</text><annotation id="Team286_A12"><infon key="type">Gene</infon><location offset="613" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A13"><infon key="type">Gene</infon><location offset="621" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>704</offset><text>Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH.</text><annotation id="Team286_A14"><infon key="type">Gene</infon><location offset="728" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A15"><infon key="type">Gene</infon><location offset="779" length="29"></location><text>plasma membrane proton-ATPase</text></annotation><annotation id="Team286_A16"><infon key="type">Gene</infon><location offset="811" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>879</offset><text>Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants.</text><annotation id="Team286_A17"><infon key="type">Gene</infon><location offset="980" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A18"><infon key="type">Gene</infon><location offset="1013" length="17"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>1033</offset><text>Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects.</text><annotation id="Team286_A19"><infon key="type">Gene</infon><location offset="1112" length="15"></location><text>Pma1p transport</text></annotation></sentence><sentence><offset>1188</offset><text>Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER.</text><annotation id="Team286_A20"><infon key="type">Gene</infon><location offset="1254" length="18"></location><text>COPII coat subunit</text></annotation><annotation id="Team286_A21"><infon key="type">Gene</infon><location offset="1279" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A22"><infon key="type">Gene</infon><location offset="1297" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A23"><infon key="type">Gene</infon><location offset="1345" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>1381</offset><sentence><offset>1381</offset><text>The plasma membrane proton-ATPase (Pma1p)1 is an essential integral membrane protein that couples ATP hydrolysis to the translocation of protons across the plasma membrane (Serrano et al., 1986).</text><annotation id="Team286_A24"><infon key="type">Gene</infon><location offset="1385" length="29"></location><text>plasma membrane proton-ATPase</text></annotation><annotation id="Team286_A25"><infon key="type">Gene</infon><location offset="1416" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>1580</offset><text>The proton gradient generated by Pma1p then drives the uptake of nutrients, such as amino acids, from the extracellular medium (Vallejo and Serrano, 1989).</text><annotation id="Team286_A26"><infon key="type">Gene</infon><location offset="1614" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>1737</offset><text>A second physiological function of Pma1p is to maintain the cytosol at a neutral pH.</text><annotation id="Team286_A27"><infon key="type">Gene</infon><location offset="1772" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>1823</offset><text>In medium of low pH, the growth rate is limited by the amount of cellular Pma1p (McCusker et al., 1987; Portillo and Serrano, 1989).</text></sentence><sentence><offset>1957</offset><text>Pma1p transport to the cell surface depends upon the secretory pathway defined by the sec genes (Brada and Schekman, 1988; Chang and Slayman, 1991).</text><annotation id="Team286_A28"><infon key="type">Gene</infon><location offset="1957" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>2109</offset><text>Pma1p is one of the most abundant cargo molecules of the secretory pathway, constituting 25-50% of the total plasma membrane protein (Serrano, 1991).</text><annotation id="Team286_A29"><infon key="type">Gene</infon><location offset="2109" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>2261</offset><text>Because of its abundance and physiological importance, one might expect that yeast cells would have specialized mechanisms to ensure efficient transport of Pma1p through the secretory pathway.</text><annotation id="Team286_A30"><infon key="type">Gene</infon><location offset="2418" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>2456</offset><text>Such a function has been suggested for two proteins, Ast1p and Ast2p, in the transport of Pma1p from the Golgi compartment to the plasma membrane (Chang and Fink, 1995).</text><annotation id="Team286_A31"><infon key="type">Gene</infon><location offset="2510" length="5"></location><text>Ast1p</text></annotation><annotation id="Team286_A32"><infon key="type">Gene</infon><location offset="2520" length="5"></location><text>Ast2p</text></annotation><annotation id="Team286_A33"><infon key="type">Gene</infon><location offset="2547" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>2629</offset><text>For early steps in the secretory pathway, proteins that are specifically required for the transport of Pma1p have not yet been identified.</text><annotation id="Team286_A34"><infon key="type">Gene</infon><location offset="2734" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>2772</offset><sentence><offset>2772</offset><text>Proteins destined for the plasma membrane are transported from the ER to the Golgi compartment by vesicles coated with a set of proteins known as COPII (Barlowe et al., 1994).</text></sentence><sentence><offset>2950</offset><text>These COPII coats are thought to cause the deformation of the membrane into a vesicle and to recruit cargo molecules into vesicle buds (reviewed by Schekman and Orci, 1996).</text><annotation id="Team286_A35"><infon key="type">Gene</infon><location offset="2956" length="11"></location><text>COPII coats</text></annotation></sentence><sentence><offset>3126</offset><text>The stepwise recruitment and assembly of the COPII coat onto the membrane is thought to occur as follows.</text></sentence><sentence><offset>3234</offset><text>Action of the ER resident membrane protein Sec12p, a guanine nucleotide exchange factor for Sar1p, causes Sar1p to bind to the ER membrane (Barlowe and Schekman, 1993).</text><annotation id="Team286_A36"><infon key="type">Gene</infon><location offset="3248" length="36"></location><text>ER resident membrane protein Sec12p</text></annotation><annotation id="Team286_A37"><infon key="type">Gene</infon><location offset="3288" length="34"></location><text>guanine nucleotide exchange factor</text></annotation><annotation id="Team286_A38"><infon key="type">Gene</infon><location offset="3327" length="5"></location><text>Sar1p</text></annotation><annotation id="Team286_A39"><infon key="type">Gene</infon><location offset="3342" length="5"></location><text>Sar1p</text></annotation></sentence><sentence><offset>3406</offset><text>Membrane-associated Sar1p, in turn, recruits the soluble Sec23p/Sec24p and Sec13p/Sec31p complexes (Matsuoka et al., 1998).</text><annotation id="Team286_A40"><infon key="type">Gene</infon><location offset="3426" length="5"></location><text>Sar1p</text></annotation><annotation id="Team286_A41"><infon key="type">Gene</infon><location offset="3463" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A42"><infon key="type">Gene</infon><location offset="3470" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A43"><infon key="type">Gene</infon><location offset="3481" length="6"></location><text>Sec13p</text></annotation><annotation id="Team286_A44"><infon key="type">Gene</infon><location offset="3488" length="6"></location><text>Sec31p</text></annotation></sentence><sentence><offset>3530</offset><text>Sec16p resides on the ER membrane and binds to both the Sec23p/Sec24p and Sec13p/Sec31p complexes, likely organizing their assembly onto the membrane (Espenshade et al., 1995; Gimeno et al., 1996; Shaywitz et al., 1997).</text><annotation id="Team286_A45"><infon key="type">Gene</infon><location offset="3530" length="6"></location><text>Sec16p</text></annotation><annotation id="Team286_A46"><infon key="type">Gene</infon><location offset="3587" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A47"><infon key="type">Gene</infon><location offset="3594" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A48"><infon key="type">Gene</infon><location offset="3606" length="6"></location><text>Sec13p</text></annotation><annotation id="Team286_A49"><infon key="type">Gene</infon><location offset="3613" length="6"></location><text>Sec31p</text></annotation></sentence><sentence><offset>3755</offset><text>To examine the role of different COPII coat subunits in recruitment of cargo molecules to vesicles, partially assembled COPII complexes have been tested for their ability to associate with cargo proteins.</text><annotation id="Team286_A50"><infon key="type">Gene</infon><location offset="3789" length="19"></location><text>COPII coat subunits</text></annotation></sentence><sentence><offset>3963</offset><text>Association of a membrane-bound complex of Sar1p and Sec23p/Sec24p with integral membrane proteins indicates that cargo proteins may laterally partition into the vesicle membrane by virtue of their affinity for the Sec23p/Sec24p protein complex (Aridor et al., 1998; Kuehn et al., 1998).</text><annotation id="Team286_A51"><infon key="type">Gene</infon><location offset="4006" length="5"></location><text>Sar1p</text></annotation><annotation id="Team286_A52"><infon key="type">Gene</infon><location offset="4017" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A53"><infon key="type">Gene</infon><location offset="4024" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A54"><infon key="type">Gene</infon><location offset="4036" length="26"></location><text>integral membrane proteins</text></annotation><annotation id="Team286_A55"><infon key="type">Gene</infon><location offset="4181" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A56"><infon key="type">Gene</infon><location offset="4188" length="6"></location><text>Sec24p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>4255</offset><sentence><offset>4255</offset><text>An early indication that the COPII coat subunits would physically interact came from specific genetic interactions between mutations in COPII genes.</text><annotation id="Team286_A57"><infon key="type">Gene</infon><location offset="4284" length="19"></location><text>COPII coat subunits</text></annotation><annotation id="Team286_A58"><infon key="type">Gene</infon><location offset="4393" length="11"></location><text>COPII genes</text></annotation></sentence><sentence><offset>4406</offset><text>When temperature-sensitive mutations in COPII genes are combined, the resulting double mutants are almost always more growth- restrictive than the component single mutations, and are usually inviable at 24 C.</text><annotation id="Team286_A59"><infon key="type">Gene</infon><location offset="4446" length="11"></location><text>COPII genes</text></annotation></sentence><sentence><offset>4616</offset><text>These synthetic-lethal interactions are restricted to genes involved in COPII vesicle formation and do not occur when mutations in genes required for vesicle formation are combined with genes required for vesicle fusion (Kaiser and Schekman, 1990).</text></sentence><sentence><offset>4867</offset><text>The specificity of this type of genetic interaction suggested that synthetic lethality with known COPII mutations would be a useful criterion to identify new mutations involved in the assembly of the COPII coat.</text><annotation id="Team286_A60"><infon key="type">Gene</infon><location offset="5069" length="10"></location><text>COPII coat</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>5082</offset><sentence><offset>5082</offset><text>We screened for mutations that were lethal with the COPII mutation sec13-1 and identified ten LST genes (lethal with sec-thirteen).</text><annotation id="Team286_A61"><infon key="type">Gene</infon><location offset="5135" length="22"></location><text>COPII mutation sec13-1</text></annotation><annotation id="Team286_A62"><infon key="type">Gene</infon><location offset="5173" length="13"></location><text>ten LST genes</text></annotation></sentence><sentence><offset>5215</offset><text>As we describe elsewhere, most of the LST genes are related to an unanticipated role for SEC13 in the regulated delivery of specific amino acid permeases to the cell surface (Roberg et al., 1997a,b).</text><annotation id="Team286_A63"><infon key="type">Gene</infon><location offset="5254" length="9"></location><text>LST genes</text></annotation><annotation id="Team286_A64"><infon key="type">Gene</infon><location offset="5306" length="5"></location><text>SEC13</text></annotation></sentence><sentence><offset>5417</offset><text>Accordingly, these LST genes display synthetic-lethal interactions with SEC13, but not with the other COPII genes.</text><annotation id="Team286_A65"><infon key="type">Gene</infon><location offset="5436" length="9"></location><text>LST genes</text></annotation><annotation id="Team286_A66"><infon key="type">Gene</infon><location offset="5489" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A67"><infon key="type">Gene</infon><location offset="5519" length="11"></location><text>COPII genes</text></annotation></sentence><sentence><offset>5533</offset><text>On the other hand, mutations in LST1 were lethal with the full set of mutations defective in COPII vesicle budding, but not with mutations defective in vesicle fusion, indicating that LST1 does participate in vesicle budding at the ER.</text><annotation id="Team286_A68"><infon key="type">Gene</infon><location offset="5565" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A69"><infon key="type">Gene</infon><location offset="5719" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>5772</offset><text>Here we show that LST1 encodes a homologue of the COPII subunit, Sec24p, and that Lst1p is a peripheral membrane protein localized to the ER that can form complexes with Sec23p.</text><annotation id="Team286_A70"><infon key="type">Gene</infon><location offset="5790" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A71"><infon key="type">Gene</infon><location offset="5823" length="13"></location><text>COPII subunit</text></annotation><annotation id="Team286_A72"><infon key="type">Gene</infon><location offset="5838" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A73"><infon key="type">Gene</infon><location offset="5855" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A74"><infon key="type">Gene</infon><location offset="5944" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>5952</offset><text>The LST1 gene is not essential, but by examination of the phenotypes of lst1Delta mutants we show that LST1 is required for the efficient export of Pma1p from the ER to the Golgi compartment.</text><annotation id="Team286_A75"><infon key="type">Gene</infon><location offset="5956" length="9"></location><text>LST1 gene</text></annotation><annotation id="Team286_A76"><infon key="type">Gene</infon><location offset="6025" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A77"><infon key="type">Gene</infon><location offset="6057" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A78"><infon key="type">Gene</infon><location offset="6102" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>6147</offset><text>These results suggest a specialized form of the vesicle coat that is responsible for recruitment of Pma1p into COPII-coated vesicles.</text><annotation id="Team286_A79"><infon key="type">Gene</infon><location offset="6247" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">title_1</infon><offset>6278</offset><sentence><offset>6278</offset><text>Materials and Methods</text></sentence></passage><passage><infon key="type">title_2</infon><offset>6300</offset><sentence><offset>6300</offset><text>Media, Strains, and Plasmids</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>6329</offset><sentence><offset>6329</offset><text>The Saccharomyces cerevisiae strains used in this study are listed in Table I.</text></sentence><sentence><offset>6409</offset><text>Rich medium (YPD) and supplemented minimal medium (SMM) were prepared according to Kaiser et al. (1994).</text></sentence><sentence><offset>6515</offset><text>To evaluate growth at low pH, YPD was adjusted to pH 3.8 with HCl (this medium remained at pH 3.8 throughout the growth of a yeast culture).</text></sentence><sentence><offset>6658</offset><text>For some experiments, SMM was buffered to pH 6.5 using 50 mM MOPS and 50 mM MES.</text></sentence><sentence><offset>6740</offset><text>Genetic manipulations were performed according to standard protocols (Kaiser et al., 1994).</text></sentence><sentence><offset>6833</offset><text>DNA manipulations were carried out as described in Sambrook et al. (1989).</text></sentence><sentence><offset>6909</offset><text>pAF70 carries the SEC24 gene in the centromere vector pCT3 (URA3; Gimeno et al., 1996).</text><annotation id="Team286_A80"><infon key="type">Gene</infon><location offset="6909" length="5"></location><text>pAF70</text></annotation><annotation id="Team286_A81"><infon key="type">Gene</infon><location offset="6927" length="10"></location><text>SEC24 gene</text></annotation></sentence><sentence><offset>6998</offset><text>pKR34 and pKR41 carry the 3.8-kb KpnI/SalI fragment containing the SEC24 gene from pAF70 in the 2mu vectors pRS426 (URA3) and pRS425 (LEU2), respectively.</text><annotation id="Team286_A82"><infon key="type">Gene</infon><location offset="6998" length="5"></location><text>pKR34</text></annotation><annotation id="Team286_A83"><infon key="type">Gene</infon><location offset="7008" length="5"></location><text>pKR41</text></annotation><annotation id="Team286_A84"><infon key="type">Gene</infon><location offset="7032" length="4"></location><text>KpnI</text></annotation><annotation id="Team286_A85"><infon key="type">Gene</infon><location offset="7037" length="4"></location><text>SalI</text></annotation><annotation id="Team286_A86"><infon key="type">Gene</infon><location offset="7066" length="10"></location><text>SEC24 gene</text></annotation><annotation id="Team286_A87"><infon key="type">Gene</infon><location offset="7082" length="5"></location><text>pAF70</text></annotation><annotation id="Team286_A88"><infon key="type">Gene</infon><location offset="7115" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A89"><infon key="type">Gene</infon><location offset="7125" length="6"></location><text>pRS425</text></annotation><annotation id="Team286_A90"><infon key="type">Gene</infon><location offset="7133" length="4"></location><text>LEU2</text></annotation></sentence><sentence><offset>7154</offset><text>pKR17 carries the LST1 gene on a 3.5-kb fragment in the centromere vector pRS316 (URA3).</text><annotation id="Team286_A91"><infon key="type">Gene</infon><location offset="7154" length="5"></location><text>pKR17</text></annotation><annotation id="Team286_A92"><infon key="type">Gene</infon><location offset="7173" length="9"></location><text>LST1 gene</text></annotation></sentence><sentence><offset>7245</offset><text>A subclone of the LST1 gene from pKR17 into the 2mu vector pRS426 gave yeast transformants at a very low efficiency because of the toxicity of LST1 sequences when present at high copy.</text><annotation id="Team286_A93"><infon key="type">Gene</infon><location offset="7263" length="9"></location><text>LST1 gene</text></annotation><annotation id="Team286_A94"><infon key="type">Gene</infon><location offset="7278" length="5"></location><text>pKR17</text></annotation><annotation id="Team286_A95"><infon key="type">Gene</infon><location offset="7390" length="14"></location><text>LST1 sequences</text></annotation></sentence><sentence><offset>7432</offset><text>To study the toxic effects of LST1, pKR35 was constructed which contains the entire LST1 coding sequence expressed from pGAL1 on pCD43 (URA3).</text><annotation id="Team286_A96"><infon key="type">Gene</infon><location offset="7463" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A97"><infon key="type">Gene</infon><location offset="7469" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A98"><infon key="type">Gene</infon><location offset="7517" length="21"></location><text>LST1 coding sequence</text></annotation><annotation id="Team286_A99"><infon key="type">Gene</infon><location offset="7554" length="5"></location><text>pGAL1</text></annotation><annotation id="Team286_A100"><infon key="type">Gene</infon><location offset="7563" length="5"></location><text>pCD43</text></annotation><annotation id="Team286_A101"><infon key="type">Gene</infon><location offset="7570" length="4"></location><text>URA3</text></annotation></sentence><sentence><offset>7577</offset><text>pKR35 will prevent growth under conditions of full induction on galactose medium, establishing that overexpression of Lst1p is toxic to yeast cells.</text><annotation id="Team286_A102"><infon key="type">Gene</infon><location offset="7577" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A103"><infon key="type">Gene</infon><location offset="7697" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>7728</offset><text>Under conditions of partial induction of pGAL1-LST1 in cells grown on raffinose, pKR35 will complement lst1Delta::LEU2 for growth on acidic medium.</text><annotation id="Team286_A104"><infon key="type">Gene</infon><location offset="7769" length="10"></location><text>pGAL1-LST1</text></annotation><annotation id="Team286_A105"><infon key="type">Gene</infon><location offset="7810" length="31"></location><text>pKR35 will complement lst1Delta</text></annotation><annotation id="Team286_A106"><infon key="type">Gene</infon><location offset="7843" length="4"></location><text>LEU2</text></annotation></sentence><sentence><offset>7877</offset><text>This shows that the LST1 open reading frame carried on pKR35 still posses LST1 function.</text><annotation id="Team286_A107"><infon key="type">Gene</infon><location offset="7898" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A108"><infon key="type">Gene</infon><location offset="7933" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A109"><infon key="type">Gene</infon><location offset="7953" length="4"></location><text>LST1</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>7968</offset><sentence><offset>7968</offset><text>Epitope-tagged LST1 was constructed as follows.</text><annotation id="Team286_A110"><infon key="type">Gene</infon><location offset="7983" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>8016</offset><text>First, the NotI site in the polylinker of pKR17 was deleted with a 350-bp SmaI/NaeI fragment (pKR17Delta), and then a 12-bp linker carrying a NotI site (1127; New England BioLabs) was inserted at the Eco47III site (at codon 13 of LST1) of pKR17Delta to make pKR17N.</text><annotation id="Team286_A111"><infon key="type">Gene</infon><location offset="8027" length="9"></location><text>NotI site</text></annotation><annotation id="Team286_A112"><infon key="type">Gene</infon><location offset="8059" length="24"></location><text>pKR17 was deleted with a</text></annotation><annotation id="Team286_A113"><infon key="type">Gene</infon><location offset="8091" length="4"></location><text>SmaI</text></annotation><annotation id="Team286_A114"><infon key="type">Gene</infon><location offset="8096" length="4"></location><text>NaeI</text></annotation><annotation id="Team286_A115"><infon key="type">Gene</infon><location offset="8112" length="10"></location><text>pKR17Delta</text></annotation><annotation id="Team286_A116"><infon key="type">Gene</infon><location offset="8160" length="9"></location><text>NotI site</text></annotation><annotation id="Team286_A117"><infon key="type">Gene</infon><location offset="8218" length="13"></location><text>Eco47III site</text></annotation><annotation id="Team286_A118"><infon key="type">Gene</infon><location offset="8258" length="10"></location><text>pKR17Delta</text></annotation><annotation id="Team286_A119"><infon key="type">Gene</infon><location offset="8277" length="6"></location><text>pKR17N</text></annotation></sentence><sentence><offset>8285</offset><text>pKR17HA carries the 100-bp NotI fragment from pGTEPI (Tyers et al., 1993), which encodes three tandem copies of the hemagglutinin (HA1) epitope, inserted into the NotI site of pKR17N.</text><annotation id="Team286_A120"><infon key="type">Gene</infon><location offset="8285" length="7"></location><text>pKR17HA</text></annotation><annotation id="Team286_A121"><infon key="type">Gene</infon><location offset="8312" length="4"></location><text>NotI</text></annotation><annotation id="Team286_A122"><infon key="type">Gene</infon><location offset="8403" length="13"></location><text>hemagglutinin</text></annotation><annotation id="Team286_A123"><infon key="type">Gene</infon><location offset="8418" length="3"></location><text>HA1</text></annotation><annotation id="Team286_A124"><infon key="type">Gene</infon><location offset="8450" length="9"></location><text>NotI site</text></annotation><annotation id="Team286_A125"><infon key="type">Gene</infon><location offset="8463" length="6"></location><text>pKR17N</text></annotation></sentence><sentence><offset>8472</offset><text>Restriction analysis using sites flanking the point of insertion revealed that two 100-bp inserts (six HA epitopes) were present in pKR17HA.</text><annotation id="Team286_A126"><infon key="type">Gene</infon><location offset="8576" length="11"></location><text>HA epitopes</text></annotation><annotation id="Team286_A127"><infon key="type">Gene</infon><location offset="8605" length="7"></location><text>pKR17HA</text></annotation></sentence><sentence><offset>8615</offset><text>pKR17HA was transformed into CKY536 to make CKY535 (MATa lst1Delta:: LEU2 leu2-3, 112 ura3-52 [pKR17HA]).</text><annotation id="Team286_A128"><infon key="type">Gene</infon><location offset="8615" length="7"></location><text>pKR17HA</text></annotation><annotation id="Team286_A129"><infon key="type">Gene</infon><location offset="8644" length="6"></location><text>CKY536</text></annotation><annotation id="Team286_A130"><infon key="type">Gene</infon><location offset="8659" length="6"></location><text>CKY535</text></annotation><annotation id="Team286_A131"><infon key="type">Gene</infon><location offset="8667" length="14"></location><text>MATa lst1Delta</text></annotation><annotation id="Team286_A132"><infon key="type">Gene</infon><location offset="8685" length="24"></location><text>LEU2 leu2-3, 112 ura3-52</text></annotation><annotation id="Team286_A133"><infon key="type">Gene</infon><location offset="8711" length="7"></location><text>pKR17HA</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>8713</offset><sentence><offset>8713</offset><text>Synthetic-lethal Screen</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>8737</offset><sentence><offset>8737</offset><text>The following plasmids and strains were constructed for use in the sec13-1 synthetic-lethal screen.</text><annotation id="Team286_A134"><infon key="type">Gene</infon><location offset="8804" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>8838</offset><text>The plasmid pKR1 carries SEC13 on a 1.8-kb SalI/ BamHI fragment excised from pCK1313 (Pryer et al., 1993), inserted into pRS316 (Sikorski and Heiter, 1989).</text><annotation id="Team286_A135"><infon key="type">Gene</infon><location offset="8863" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A136"><infon key="type">Gene</infon><location offset="8881" length="4"></location><text>SalI</text></annotation><annotation id="Team286_A137"><infon key="type">Gene</infon><location offset="8887" length="5"></location><text>BamHI</text></annotation></sentence><sentence><offset>8996</offset><text>pKR4 carries the same 1.8-kb SalI/ BamHI fragment and a 3.8-kb NheI/BamHI fragment containing ADE3 from pDK255, both inserted into the vector pRS315 (Sikorski and Heiter, 1989).</text><annotation id="Team286_A138"><infon key="type">Gene</infon><location offset="8996" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A139"><infon key="type">Gene</infon><location offset="9025" length="4"></location><text>SalI</text></annotation><annotation id="Team286_A140"><infon key="type">Gene</infon><location offset="9031" length="5"></location><text>BamHI</text></annotation><annotation id="Team286_A141"><infon key="type">Gene</infon><location offset="9059" length="4"></location><text>NheI</text></annotation><annotation id="Team286_A142"><infon key="type">Gene</infon><location offset="9064" length="5"></location><text>BamHI</text></annotation><annotation id="Team286_A143"><infon key="type">Gene</infon><location offset="9090" length="4"></location><text>ADE3</text></annotation></sentence><sentence><offset>9176</offset><text>CUY563 and CKY45 were crossed to produce a MATa ade2 ade3 leu2 ura3 sec13-1 segregant, which was transformed with pKR4 to give CKY423.</text><annotation id="Team286_A144"><infon key="type">Gene</infon><location offset="9176" length="6"></location><text>CUY563</text></annotation><annotation id="Team286_A145"><infon key="type">Gene</infon><location offset="9187" length="5"></location><text>CKY45</text></annotation><annotation id="Team286_A146"><infon key="type">Gene</infon><location offset="9219" length="4"></location><text>MATa</text></annotation><annotation id="Team286_A147"><infon key="type">Gene</infon><location offset="9224" length="4"></location><text>ade2</text></annotation><annotation id="Team286_A148"><infon key="type">Gene</infon><location offset="9229" length="4"></location><text>ade3</text></annotation><annotation id="Team286_A149"><infon key="type">Gene</infon><location offset="9235" length="4"></location><text>leu2</text></annotation><annotation id="Team286_A150"><infon key="type">Gene</infon><location offset="9240" length="22"></location><text>ura3 sec13-1 segregant</text></annotation><annotation id="Team286_A151"><infon key="type">Gene</infon><location offset="9291" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A152"><infon key="type">Gene</infon><location offset="9305" length="6"></location><text>CKY423</text></annotation></sentence><sentence><offset>9313</offset><text>The mating type of CKY423 was switched by ectopic expression of the HO gene (Herskowitz and Jensen, 1991) to give CKY424.</text><annotation id="Team286_A153"><infon key="type">Gene</infon><location offset="9332" length="6"></location><text>CKY423</text></annotation><annotation id="Team286_A154"><infon key="type">Gene</infon><location offset="9382" length="7"></location><text>HO gene</text></annotation><annotation id="Team286_A155"><infon key="type">Gene</infon><location offset="9428" length="6"></location><text>CKY424</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>9436</offset><sentence><offset>9436</offset><text>Cultures of CKY423 and CKY424 were mutagenized by irradiation with a germicidal UV lamp at a dose resulting in 10% cell survival.</text><annotation id="Team286_A156"><infon key="type">Gene</infon><location offset="9448" length="6"></location><text>CKY423</text></annotation><annotation id="Team286_A157"><infon key="type">Gene</infon><location offset="9459" length="6"></location><text>CKY424</text></annotation></sentence><sentence><offset>9567</offset><text>Mutagenized cells were plated on YPD at a density of 150 colonies per plate.</text></sentence><sentence><offset>9645</offset><text>After 5 d of growth at 24 C, colonies with a solid red color and no white sectors were selected for further analysis.</text></sentence><sentence><offset>9764</offset><text>The dependence of the nonsectoring phenotype on the sec13-1 mutation was tested by transforming candidate mutants with either pKR1 or pRS316.</text><annotation id="Team286_A158"><infon key="type">Gene</infon><location offset="9816" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>9906</offset><text>Strains that sectored after transformation with pKR1, but not after transformation with pRS316, were scored as sec13-1-dependent.</text><annotation id="Team286_A159"><infon key="type">Gene</infon><location offset="9955" length="4"></location><text>pKR1</text></annotation><annotation id="Team286_A160"><infon key="type">Gene</infon><location offset="9995" length="6"></location><text>pRS316</text></annotation><annotation id="Team286_A161"><infon key="type">Gene</infon><location offset="10019" length="7"></location><text>sec13-1</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>10041</offset><sentence><offset>10041</offset><text>Complementation tests were performed by mating mutants isolated from CKY423 with those isolated from CKY424.</text><annotation id="Team286_A162"><infon key="type">Gene</infon><location offset="10111" length="6"></location><text>CKY423</text></annotation><annotation id="Team286_A163"><infon key="type">Gene</infon><location offset="10143" length="6"></location><text>CKY424</text></annotation></sentence><sentence><offset>10151</offset><text>Zygotes isolated by micromanipulation were scored for their ability to form sectored colonies on YPD plates.</text></sentence><sentence><offset>10261</offset><text>The genes defined by these complementation groups were designated LST.</text><annotation id="Team286_A164"><infon key="type">Gene</infon><location offset="10328" length="3"></location><text>LST</text></annotation></sentence><sentence><offset>10333</offset><text>All lst mutant strains were backcrossed to a parental strain twice.</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>10402</offset><sentence><offset>10402</offset><text>The lst sec13-1 double mutants were converted to lst single mutants by integration of a wild-type copy of SEC13 at the sec13-1 locus, using the integrating plasmid p1312 (SEC13 URA3; Pryer et al., 1993).</text><annotation id="Team286_A165"><infon key="type">Gene</infon><location offset="10410" length="22"></location><text>sec13-1 double mutants</text></annotation><annotation id="Team286_A166"><infon key="type">Gene</infon><location offset="10509" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A167"><infon key="type">Gene</infon><location offset="10522" length="13"></location><text>sec13-1 locus</text></annotation><annotation id="Team286_A168"><infon key="type">Gene</infon><location offset="10567" length="5"></location><text>p1312</text></annotation><annotation id="Team286_A169"><infon key="type">Gene</infon><location offset="10574" length="10"></location><text>SEC13 URA3</text></annotation></sentence><sentence><offset>10607</offset><text>The integrants were grown on YPD and cells from white sectors (indicating loss of pKR4) were isolated.</text><annotation id="Team286_A170"><infon key="type">Gene</infon><location offset="10691" length="4"></location><text>pKR4</text></annotation></sentence><sentence><offset>10712</offset><text>The integration of a wild-type copy of SEC13 was confirmed by the ability of the cells from white sectors to grow at 36 C, a temperature that is restrictive for the sec13-1 mutation.</text><annotation id="Team286_A171"><infon key="type">Gene</infon><location offset="10751" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A172"><infon key="type">Gene</infon><location offset="10879" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>10897</offset><text>Owing to the poor growth of lst9 strains, we were not able to construct a lst9 single mutant by this method.</text><annotation id="Team286_A173"><infon key="type">Gene</infon><location offset="10926" length="4"></location><text>lst9</text></annotation><annotation id="Team286_A174"><infon key="type">Gene</infon><location offset="10972" length="18"></location><text>lst9 single mutant</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>11009</offset><sentence><offset>11009</offset><text>To test for synthetic-lethal interactions between lst mutations and mutations in sec genes, lst mutants CKY435 (lst1-1), CKY436 (lst2-1), CKY437 (lst3-1), CKY438 (lst4-1), CKY439 (lst5-1), CKY440 (lst6-1), CKY441 (lst7-1), and CKY442 (lst8-1) were crossed to the sec mutants CKY45 (sec13-1), CKY50 (sec16-2), CKY78 (sec23-1), and CKY450 (sec31-2).</text><annotation id="Team286_A175"><infon key="type">Gene</infon><location offset="11101" length="18"></location><text>lst mutants CKY435</text></annotation><annotation id="Team286_A176"><infon key="type">Gene</infon><location offset="11121" length="6"></location><text>lst1-1</text></annotation><annotation id="Team286_A177"><infon key="type">Gene</infon><location offset="11130" length="6"></location><text>CKY436</text></annotation><annotation id="Team286_A178"><infon key="type">Gene</infon><location offset="11138" length="6"></location><text>lst2-1</text></annotation><annotation id="Team286_A179"><infon key="type">Gene</infon><location offset="11147" length="6"></location><text>CKY437</text></annotation><annotation id="Team286_A180"><infon key="type">Gene</infon><location offset="11156" length="6"></location><text>lst3-1</text></annotation><annotation id="Team286_A181"><infon key="type">Gene</infon><location offset="11165" length="6"></location><text>CKY438</text></annotation><annotation id="Team286_A182"><infon key="type">Gene</infon><location offset="11173" length="6"></location><text>lst4-1</text></annotation><annotation id="Team286_A183"><infon key="type">Gene</infon><location offset="11182" length="6"></location><text>CKY439</text></annotation><annotation id="Team286_A184"><infon key="type">Gene</infon><location offset="11190" length="6"></location><text>lst5-1</text></annotation><annotation id="Team286_A185"><infon key="type">Gene</infon><location offset="11199" length="6"></location><text>CKY440</text></annotation><annotation id="Team286_A186"><infon key="type">Gene</infon><location offset="11207" length="6"></location><text>lst6-1</text></annotation><annotation id="Team286_A187"><infon key="type">Gene</infon><location offset="11216" length="6"></location><text>CKY441</text></annotation><annotation id="Team286_A188"><infon key="type">Gene</infon><location offset="11225" length="6"></location><text>lst7-1</text></annotation><annotation id="Team286_A189"><infon key="type">Gene</infon><location offset="11238" length="6"></location><text>CKY442</text></annotation><annotation id="Team286_A190"><infon key="type">Gene</infon><location offset="11246" length="6"></location><text>lst8-1</text></annotation><annotation id="Team286_A191"><infon key="type">Gene</infon><location offset="11274" length="17"></location><text>sec mutants CKY45</text></annotation><annotation id="Team286_A192"><infon key="type">Gene</infon><location offset="11294" length="7"></location><text>sec13-1</text></annotation><annotation id="Team286_A193"><infon key="type">Gene</infon><location offset="11304" length="5"></location><text>CKY50</text></annotation><annotation id="Team286_A194"><infon key="type">Gene</infon><location offset="11311" length="7"></location><text>sec16-2</text></annotation><annotation id="Team286_A195"><infon key="type">Gene</infon><location offset="11321" length="5"></location><text>CKY78</text></annotation><annotation id="Team286_A196"><infon key="type">Gene</infon><location offset="11328" length="7"></location><text>sec23-1</text></annotation><annotation id="Team286_A197"><infon key="type">Gene</infon><location offset="11342" length="6"></location><text>CKY450</text></annotation><annotation id="Team286_A198"><infon key="type">Gene</infon><location offset="11350" length="7"></location><text>sec31-2</text></annotation></sentence><sentence><offset>11360</offset><text>Inviability of a given lst sec double mutant was inferred from crosses where lethality segregated as a two-gene trait (most tetrads giving a segregation pattern of 1:3 for lethality), an outcome that was easily detectable since crosses to wild-type typically gave &gt;95% spore viability.</text><annotation id="Team286_A199"><infon key="type">Gene</infon><location offset="11383" length="21"></location><text>lst sec double mutant</text></annotation></sentence><sentence><offset>11649</offset><text>The segregation pattern of the sec mutation in the surviving sister spores was used as an additional test to establish that the inviable spores always carried the sec mutation, and therefore were not the result of random spore death.</text></sentence></passage><passage><infon key="type">title_2</infon><offset>11884</offset><sentence><offset>11884</offset><text>Construction of lst1Delta Mutants</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>11915</offset><sentence><offset>11915</offset><text>Replacement of the chromosomal LST1 gene with an allele disrupted with the LEU2 gene was constructed as follows.</text><annotation id="Team286_A200"><infon key="type">Gene</infon><location offset="11934" length="21"></location><text>chromosomal LST1 gene</text></annotation><annotation id="Team286_A201"><infon key="type">Gene</infon><location offset="11991" length="9"></location><text>LEU2 gene</text></annotation></sentence><sentence><offset>12029</offset><text>pKR18 carries the 5' half of LST1 on a 2.0-kb Xho1/HindIII fragment inserted into pRS316.</text><annotation id="Team286_A202"><infon key="type">Gene</infon><location offset="12029" length="5"></location><text>pKR18</text></annotation><annotation id="Team286_A203"><infon key="type">Gene</infon><location offset="12059" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A204"><infon key="type">Gene</infon><location offset="12076" length="4"></location><text>Xho1</text></annotation><annotation id="Team286_A205"><infon key="type">Gene</infon><location offset="12081" length="7"></location><text>HindIII</text></annotation></sentence><sentence><offset>12120</offset><text>A 2.0-kb HindIII/BamHI fragment containing the LEU2 gene from plasmid pJJ252 (Jones and Prakash, 1990) and a 250-bp BclI/SacI fragment from the 3' noncoding region of LST1 were inserted into pKR18 to construct pKR28.</text><annotation id="Team286_A206"><infon key="type">Gene</infon><location offset="12130" length="7"></location><text>HindIII</text></annotation><annotation id="Team286_A207"><infon key="type">Gene</infon><location offset="12138" length="5"></location><text>BamHI</text></annotation><annotation id="Team286_A208"><infon key="type">Gene</infon><location offset="12168" length="9"></location><text>LEU2 gene</text></annotation><annotation id="Team286_A209"><infon key="type">Gene</infon><location offset="12238" length="4"></location><text>BclI</text></annotation><annotation id="Team286_A210"><infon key="type">Gene</infon><location offset="12243" length="4"></location><text>SacI</text></annotation><annotation id="Team286_A211"><infon key="type">Gene</infon><location offset="12290" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A212"><infon key="type">Gene</infon><location offset="12314" length="5"></location><text>pKR18</text></annotation></sentence><sentence><offset>12341</offset><text>The NH2-terminal coding region of LST1 (except for codons 1-13) was removed by deleting a 1.7-kb Eco47III/MscI fragment from pKR28 to generate pKR28Delta.</text><annotation id="Team286_A213"><infon key="type">Gene</infon><location offset="12375" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A214"><infon key="type">Gene</infon><location offset="12438" length="8"></location><text>Eco47III</text></annotation><annotation id="Team286_A215"><infon key="type">Gene</infon><location offset="12447" length="4"></location><text>MscI</text></annotation><annotation id="Team286_A216"><infon key="type">Gene</infon><location offset="12466" length="5"></location><text>pKR28</text></annotation><annotation id="Team286_A217"><infon key="type">Gene</infon><location offset="12484" length="10"></location><text>pKR28Delta</text></annotation></sentence><sentence><offset>12496</offset><text>The lst1Delta::LEU2 construct, liberated from pKR28Delta by digestion with XhoI, was transformed into the wild-type diploid strain CKY348 (MATa/alpha leu2-3,112/leu2-3,112 ura3-52/ura3-52).</text><annotation id="Team286_A218"><infon key="type">Gene</infon><location offset="12511" length="14"></location><text>LEU2 construct</text></annotation><annotation id="Team286_A219"><infon key="type">Gene</infon><location offset="12542" length="10"></location><text>pKR28Delta</text></annotation><annotation id="Team286_A220"><infon key="type">Gene</infon><location offset="12571" length="4"></location><text>XhoI</text></annotation><annotation id="Team286_A221"><infon key="type">Gene</infon><location offset="12628" length="6"></location><text>CKY348</text></annotation><annotation id="Team286_A222"><infon key="type">Gene</infon><location offset="12636" length="28"></location><text>MATa/alpha leu2-3,112/leu2-3</text></annotation><annotation id="Team286_A223"><infon key="type">Gene</infon><location offset="12665" length="3"></location><text>112</text></annotation><annotation id="Team286_A224"><infon key="type">Gene</infon><location offset="12669" length="7"></location><text>ura3-52</text></annotation><annotation id="Team286_A225"><infon key="type">Gene</infon><location offset="12677" length="7"></location><text>ura3-52</text></annotation></sentence><sentence><offset>12687</offset><text>On sporulation and dissection, this diploid gave four viable spore clones, and haploid segregants carrying lst1Delta::LEU2 were confirmed by Southern blotting.</text><annotation id="Team286_A226"><infon key="type">Gene</infon><location offset="12795" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A227"><infon key="type">Gene</infon><location offset="12806" length="4"></location><text>LEU2</text></annotation></sentence><sentence><offset>12848</offset><text>One such segregant was further backcrossed to our S288C genetic background to give strains CKY536 and CKY542.</text><annotation id="Team286_A228"><infon key="type">Gene</infon><location offset="12952" length="6"></location><text>CKY542</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>12951</offset><sentence><offset>12951</offset><text>Proton Efflux from Intact Yeast Cells</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>12989</offset><sentence><offset>12989</offset><text>Pma1p activity was assayed by proton efflux from intact cells into the external medium.</text><annotation id="Team286_A229"><infon key="type">Gene</infon><location offset="12989" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>13077</offset><text>Cells were grown to exponential phase in YPD at 37 C, washed, and then stored in deionized water at 4 C overnight.</text></sentence><sentence><offset>13193</offset><text>Cell number was measured by light scattering, and a total of 25 A600 units (~5 x 108 cells) was suspended in 5 ml of 100 mM KCl, 10 mM glycine, pH 4.0.</text></sentence><sentence><offset>13347</offset><text>The pH of the cell suspension was measured using a combination electrode at 25 C with constant stirring.</text></sentence><sentence><offset>13454</offset><text>Once the pH had stabilized (~10 min), glucose was added to a final concentration of 40 mM and the ensuing drop in pH was recorded at 30-s intervals over 15 min.</text></sentence><sentence><offset>13616</offset><text>In comparison of wild-type (CKY443) and lst1Delta (CKY536) strains, both suspensions had identical cell concentration as measured by light scattering, and showed the same response to calibration pulses with HCl.</text><annotation id="Team286_A230"><infon key="type">Gene</infon><location offset="13633" length="19"></location><text>wild-type (CKY443)</text></annotation><annotation id="Team286_A231"><infon key="type">Gene</infon><location offset="13657" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A232"><infon key="type">Gene</infon><location offset="13668" length="6"></location><text>CKY536</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>13835</offset><sentence><offset>13835</offset><text>Immunofluorescence Microscopy</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>13865</offset><sentence><offset>13865</offset><text>The intracellular location of Pma1p in wild-type (CKY443) and lst1Delta (CKY536) cells was examined by indirect immunofluorescence microscopy using techniques described previously (Pringle et al., 1991; Espenshade et al., 1995).</text><annotation id="Team286_A233"><infon key="type">Gene</infon><location offset="13895" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A234"><infon key="type">Gene</infon><location offset="13927" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A235"><infon key="type">Gene</infon><location offset="13939" length="6"></location><text>CKY536</text></annotation></sentence><sentence><offset>14095</offset><text>Strains were grown exponentially in SMM medium, pH 7.2, at 30 C.</text></sentence><sentence><offset>14161</offset><text>Cells were fixed in 3.7% formaldehyde and then converted to spheroplasts by digestion with lyticase.</text><annotation id="Team286_A236"><infon key="type">Gene</infon><location offset="14253" length="8"></location><text>lyticase</text></annotation></sentence><sentence><offset>14263</offset><text>Both primary and secondary antibody incubations were for 1 h at 25 C.</text></sentence><sentence><offset>14333</offset><text>Affinity-purified anti-Pma1p antibody was prepared as follows.</text><annotation id="Team286_A237"><infon key="type">Gene</infon><location offset="14351" length="19"></location><text>anti-Pma1p antibody</text></annotation></sentence><sentence><offset>14396</offset><text>A crude preparation of yeast membranes was resolved by preparative SDS-PAGE, and after transfer of proteins to a nitrocellulose membrane by electrophoresis, the strip of membrane that contained Pma1p was excised.</text><annotation id="Team286_A238"><infon key="type">Gene</infon><location offset="14593" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>14612</offset><text>Rabbit antiserum to Pma1p was applied to the nitrocellulose strip, and after the strip was washed with 20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween 20, the bound antibody was eluted with 100 mM glycine, pH 2.8, 500 mM NaCl, 0.5% Tween 20.</text><annotation id="Team286_A239"><infon key="type">Gene</infon><location offset="14632" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>14853</offset><text>Affinity-purified Pma1p was used at a 1:100 dilution and FITC-conjugated anti-rabbit IgG was used at 1:200 dilution.</text><annotation id="Team286_A240"><infon key="type">Gene</infon><location offset="14871" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A241"><infon key="type">Gene</infon><location offset="14926" length="16"></location><text>anti-rabbit IgG</text></annotation></sentence><sentence><offset>14971</offset><text>Mounting medium was supplemented with 4',6-diamidino-2-phenylindole (DAPI).</text></sentence><sentence><offset>15048</offset><text>Micrographs were taken with a Nikon Eclipse TE300 microscope with a Hamamatsu Orca C4742-95 CCD camera.</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>15155</offset><sentence><offset>15155</offset><text>For the localization of Lst1p-HA, CKY535 was grown on SMM to exponential phase and prepared as described above.</text><annotation id="Team286_A242"><infon key="type">Gene</infon><location offset="15179" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A243"><infon key="type">Gene</infon><location offset="15189" length="6"></location><text>CKY535</text></annotation></sentence><sentence><offset>15267</offset><text>For visualization of Lst1p-HA, the 12CA5 antibody (Berkeley Antibody Co., Inc.) was used at a 1:5,000 dilution and FITC-conjugated goat anti-mouse IgG was used at a 1:50 dilution.</text><annotation id="Team286_A244"><infon key="type">Gene</infon><location offset="15289" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A245"><infon key="type">Gene</infon><location offset="15405" length="14"></location><text>anti-mouse IgG</text></annotation></sentence><sentence><offset>15450</offset><text>Rabbit anti-Kar2p polyclonal serum (a gift of M. Rose, Princeton University, Princeton, NJ) was used at a 1:1,000 dilution and rhodamine-conjugated goat anti-rabbit IgG was used at a 1:200 dilution.</text><annotation id="Team286_A246"><infon key="type">Gene</infon><location offset="15450" length="17"></location><text>Rabbit anti-Kar2p</text></annotation><annotation id="Team286_A247"><infon key="type">Gene</infon><location offset="15605" length="15"></location><text>anti-rabbit IgG</text></annotation></sentence><sentence><offset>15652</offset><text>Samples were viewed and imaged using a Nikon Optiphot 2 microscope and a Photometric ImagePoint CCD camera.</text></sentence><sentence><offset>15761</offset><text>Images were recorded using IP-Lab software (Molecular Dynamics, Inc.).</text></sentence></passage><passage><infon key="type">title_2</infon><offset>15839</offset><sentence><offset>15839</offset><text>Cell Fractionation</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>15858</offset><sentence><offset>15858</offset><text>Cell organelles were fractionated on equilibrium density gradients as previously described (Roberg et al., 1997a).</text></sentence><sentence><offset>15973</offset><text>Cultures were grown exponentially at 24 C and then shifted to 37 C for 3 h.</text></sentence><sentence><offset>16049</offset><text>1.6 x 109 cells were collected by centrifugation and suspended in 0.5 ml STE10 (10% [wt/wt] sucrose, 10 mM Tris-HCl, pH 7.6, 10 mM EDTA) with a protease inhibitor cocktail (1 mM PMSF, 0.5 mug/ml leupeptin, 0.7 mug/ml pepstatin, 2 mug/ml aprotinin) and lysed by vortexing with glass beads.</text><annotation id="Team286_A248"><infon key="type">Gene</infon><location offset="16268" length="29"></location><text>pepstatin, 2 mug/ml aprotinin</text></annotation></sentence><sentence><offset>16340</offset><text>An additional 1 ml of STE10 was added, and the lysate was cleared of unbroken cells and large cell debris by centrifugation at 300 g for 2 min.</text><annotation id="Team286_A249"><infon key="type">Gene</infon><location offset="16362" length="5"></location><text>STE10</text></annotation></sentence><sentence><offset>16485</offset><text>The cleared extract (300 mul) was layered on top of a 5-ml, 20-60% linear sucrose gradient in TE (10 mM Tris-HCl, pH 7.6, 10 mM EDTA) prepared for an SW50.1 rotor (Beckman Instruments, Inc.).</text></sentence><sentence><offset>16679</offset><text>Samples were centrifuged 100,000 g for 18 h at 4 C and fractions of 300 mul were collected from the top of the gradient.</text></sentence><sentence><offset>16801</offset><text>Protein was precipitated from each fraction by the addition of 100 mul of 0.15% deoxycholate and 100 mul of 72% trichloroacetic acid.</text></sentence><sentence><offset>16937</offset><text>Protein pellets were collected by centrifugation at 13,000 g, washed with cold acetone, and then solubilized in ESB (60 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.02% bromophenol blue).</text></sentence><sentence><offset>17140</offset><text>Pma1p, Gas1p, and Sec61p were resolved by SDS-PAGE and were detected by immunoblotting.</text><annotation id="Team286_A250"><infon key="type">Gene</infon><location offset="17140" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A251"><infon key="type">Gene</infon><location offset="17147" length="5"></location><text>Gas1p</text></annotation><annotation id="Team286_A252"><infon key="type">Gene</infon><location offset="17158" length="6"></location><text>Sec61p</text></annotation></sentence><sentence><offset>17229</offset><text>The relative amount of each protein in cell fractions was determined by densitometry using an Ultroscan 2202 (LKB Instruments, Inc.).</text></sentence><sentence><offset>17363</offset><text>The Golgi GDPase activity was assayed in gradient fractions before protein precipitation using standard methods (Abeijon et al., 1989).</text><annotation id="Team286_A253"><infon key="type">Gene</infon><location offset="17367" length="12"></location><text>Golgi GDPase</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>17506</offset><sentence><offset>17506</offset><text>The subcellular distribution of Lst1p-HA was examined using techniques described previously (Espenshade et al., 1995).</text><annotation id="Team286_A254"><infon key="type">Gene</infon><location offset="17538" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>17625</offset><text>CKY535 carrying pKR17HA, which expresses Lst1p-HA, was grown to exponential phase in SMM without uracil.</text><annotation id="Team286_A255"><infon key="type">Gene</infon><location offset="17625" length="6"></location><text>CKY535</text></annotation><annotation id="Team286_A256"><infon key="type">Gene</infon><location offset="17642" length="7"></location><text>pKR17HA</text></annotation><annotation id="Team286_A257"><infon key="type">Gene</infon><location offset="17667" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>17732</offset><text>2 x 109 cells were harvested, converted to spheroplasts, and then gently lysed by glass beads in 500 mul of cell lysis buffer (20 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl2) including the protease inhibitor cocktail.</text></sentence><sentence><offset>17948</offset><text>The cell extract was sequentially centrifuged at 500 g for 20 min, 10,000 g for 20 min, and 150,000 g for 60 min, to give one soluble and three particulate fractions.</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>18117</offset><sentence><offset>18117</offset><text>Release of Lst1p-HA from the particulate fraction was examined by treating cell extracts with 500 mM NaCl, 100 mM sodium carbonate, pH 11.5, 2.5 M urea, or 1% Triton X-100.</text><annotation id="Team286_A258"><infon key="type">Gene</infon><location offset="18117" length="7"></location><text>Release</text></annotation><annotation id="Team286_A259"><infon key="type">Gene</infon><location offset="18128" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>18292</offset><text>After 1 h of incubation at 4 C, samples were centrifuged at 50,000 g for 30 min to separate soluble and particulate fractions.</text></sentence><sentence><offset>18419</offset><text>Fractions from both experiments were solubilized in sample buffer and analyzed by immunoblotting.</text></sentence></passage><passage><infon key="type">title_2</infon><offset>18518</offset><sentence><offset>18518</offset><text>Immunoblotting</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>18533</offset><sentence><offset>18533</offset><text>Samples of 10-30 mul in ESB were resolved by SDS-PAGE and immunoblotting was conducted according to standard protocols (Harlow and Lane, 1988).</text></sentence><sentence><offset>18678</offset><text>For transfer of Lst1p to nitrocellulose membranes, 0.1% SDS was included in the transfer buffer.</text><annotation id="Team286_A260"><infon key="type">Gene</infon><location offset="18694" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>18776</offset><text>The following antibodies were used: mouse monoclonal 12CA5 anti-HA at 1:1,000 dilution; rabbit anti-Pma1p (a gift of A. Chang, Albert Einstein College of Medicine, Bronx, NY) at 1:500 dilution; rabbit anti-Gas1p (a gift of H. Riezman, University of Basel, Basel, Switzerland) at 1:10,000 dilution; rabbit anti-Sec61p (a gift of R. Schekman, University of California, Berkeley, CA) at 1:3,000 dilution; rabbit anti-Gdh2p (a gift of B. Magasanik, Massachusetts Institute of Technology, Cambridge, MA) at 1:1,000 dilution; HRP-coupled sheep anti-mouse Ig and HRP-coupled sheep anti-rabbit Ig (Nycomed Amersham Corp.) at 1:10,000 dilution.</text><annotation id="Team286_A261"><infon key="type">Gene</infon><location offset="18813" length="30"></location><text>mouse monoclonal 12CA5 anti-HA</text></annotation><annotation id="Team286_A262"><infon key="type">Gene</infon><location offset="18865" length="17"></location><text>rabbit anti-Pma1p</text></annotation><annotation id="Team286_A263"><infon key="type">Gene</infon><location offset="18973" length="17"></location><text>rabbit anti-Gas1p</text></annotation><annotation id="Team286_A264"><infon key="type">Gene</infon><location offset="19078" length="18"></location><text>rabbit anti-Sec61p</text></annotation><annotation id="Team286_A265"><infon key="type">Gene</infon><location offset="19184" length="17"></location><text>rabbit anti-Gdh2p</text></annotation><annotation id="Team286_A266"><infon key="type">Gene</infon><location offset="19303" length="69"></location><text>HRP-coupled sheep anti-mouse Ig and HRP-coupled sheep anti-rabbit Ig</text></annotation></sentence><sentence><offset>19420</offset><text>Blots were developed using chemiluminescence detection system (Nycomed Amersham Corp.).</text></sentence></passage><passage><infon key="type">title_2</infon><offset>19514</offset><sentence><offset>19514</offset><text>Pulse-Chase Kinetics of Invertase Maturation</text><annotation id="Team286_A267"><infon key="type">Gene</infon><location offset="19538" length="9"></location><text>Invertase</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>19561</offset><sentence><offset>19561</offset><text>The strains used for radiolabeling all carried the plasmid pNV31, which carries the SUC2 gene under the constitutive TPI1 promoter (a gift of M. Lewis, Medical Research Council Laboratories of Molecular Biology, Cambridge, UK).</text><annotation id="Team286_A268"><infon key="type">Gene</infon><location offset="19646" length="9"></location><text>SUC2 gene</text></annotation><annotation id="Team286_A269"><infon key="type">Gene</infon><location offset="19679" length="13"></location><text>TPI1 promoter</text></annotation></sentence><sentence><offset>19792</offset><text>Wild-type (CKY540) and lst1Delta (CKY542) strains were grown in SMM without methionine (buffered with 50 mM MES and 50 mM MOPS to pH 6.5) at 24 C to exponential phase, and then shifted to 37 C for 3 h before labeling.</text><annotation id="Team286_A270"><infon key="type">Gene</infon><location offset="19792" length="18"></location><text>Wild-type (CKY540)</text></annotation><annotation id="Team286_A271"><infon key="type">Gene</infon><location offset="19815" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A272"><infon key="type">Gene</infon><location offset="19826" length="6"></location><text>CKY542</text></annotation></sentence><sentence><offset>20013</offset><text>A sec12-4 strain (CKY541) was similarly grown to exponential phase at 24 C, but was shifted to 37 C 5 min before the addition of label.</text><annotation id="Team286_A273"><infon key="type">Gene</infon><location offset="20015" length="14"></location><text>sec12-4 strain</text></annotation><annotation id="Team286_A274"><infon key="type">Gene</infon><location offset="20031" length="6"></location><text>CKY541</text></annotation></sentence><sentence><offset>20150</offset><text>Radiolabeling and immunoprecipitation of invertase was performed as previously described (Gimeno et al., 1995; Elrod-Erickson and Kaiser, 1996).</text><annotation id="Team286_A275"><infon key="type">Gene</infon><location offset="20191" length="9"></location><text>invertase</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>20298</offset><sentence><offset>20298</offset><text>Two-Hybrid Interactions</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>20322</offset><sentence><offset>20322</offset><text>The yeast two-hybrid assay was used to test potential protein-protein interactions as previously described (Gyuris et al., 1993; Bartel and Fields, 1995).</text></sentence><sentence><offset>20478</offset><text>Interactions were tested between either Lst1p or Sec24p fused to the LexA DNA-binding domain and Sec23p fused to an acidic transcriptional activation domain.</text><annotation id="Team286_A276"><infon key="type">Gene</infon><location offset="20518" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A277"><infon key="type">Gene</infon><location offset="20527" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A278"><infon key="type">Gene</infon><location offset="20548" length="23"></location><text>LexA DNA-binding domain</text></annotation><annotation id="Team286_A279"><infon key="type">Gene</infon><location offset="20576" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>20637</offset><text>The following plasmids were used: pPE81 carries SEC23 fused to the acidic activation domain of pJG4-5 (Espenshade et al., 1995); pRH286 carries SEC24 (codons 34-926) fused to the lexA DNA-binding domain in pEG202 (Gimeno et al., 1996); pKR37 carries LST1 fused to the lexA DNA-binding domain in pGilda (a derivative of pEG202 with pGAL1; provided by D. Shaywitz).</text><annotation id="Team286_A280"><infon key="type">Gene</infon><location offset="20671" length="5"></location><text>pPE81</text></annotation><annotation id="Team286_A281"><infon key="type">Gene</infon><location offset="20685" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A282"><infon key="type">Gene</infon><location offset="20732" length="6"></location><text>pJG4-5</text></annotation><annotation id="Team286_A283"><infon key="type">Gene</infon><location offset="20782" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A284"><infon key="type">Gene</infon><location offset="20817" length="24"></location><text>lexA DNA-binding domain</text></annotation><annotation id="Team286_A285"><infon key="type">Gene</infon><location offset="20875" length="5"></location><text>pKR37</text></annotation><annotation id="Team286_A286"><infon key="type">Gene</infon><location offset="20890" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A287"><infon key="type">Gene</infon><location offset="20908" length="23"></location><text>lexA DNA-binding domain</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>21009</offset><sentence><offset>21009</offset><text>Combinations of control and fusion protein plasmids, along with the reporter plasmid pSH18-34, were transformed into the strain EGY40 (Golemis and Brent, 1992).</text></sentence><sentence><offset>21170</offset><text>Strains were grown exponentially in SMM with 2% raffinose as the carbon source.</text></sentence><sentence><offset>21251</offset><text>Galactose was added to a concentration of 2%, and incubation was continued for 10 h to induce fusion proteins expressed from pGAL1.</text><annotation id="Team286_A288"><infon key="type">Gene</infon><location offset="21377" length="5"></location><text>pGAL1</text></annotation></sentence><sentence><offset>21384</offset><text>Assays for beta-galactosidase activity were performed on cells lysed by disruption with glass beads (Rose and Botstein, 1983).</text><annotation id="Team286_A289"><infon key="type">Gene</infon><location offset="21395" length="18"></location><text>beta-galactosidase</text></annotation></sentence><sentence><offset>21513</offset><text>Activity was normalized to total protein determined by the Bradford assay (Bio-Rad Laboratories).</text></sentence></passage><passage><infon key="type">title_2</infon><offset>21609</offset><sentence><offset>21609</offset><text>Binding of Lst1p to Sec23p</text><annotation id="Team286_A290"><infon key="type">Gene</infon><location offset="21620" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A291"><infon key="type">Gene</infon><location offset="21629" length="6"></location><text>Sec23p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>21636</offset><sentence><offset>21636</offset><text>A gene fusion expressing Lst1p fused to glutathione S-transferase (GST) was constructed by inserting the 3.0-kb BamHI/XhoI fragment of pKR17HA into pRD56 (a gift of R. Deshaies, California Institute of Technology, Pasadena, CA) to construct pRH254, which gives GST-Lst1p-HA (amino acids 14-927 of Lst1p) fusion expressed from pGAL1.</text><annotation id="Team286_A292"><infon key="type">Gene</infon><location offset="21638" length="11"></location><text>gene fusion</text></annotation><annotation id="Team286_A293"><infon key="type">Gene</infon><location offset="21661" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A294"><infon key="type">Gene</infon><location offset="21676" length="11"></location><text>glutathione</text></annotation><annotation id="Team286_A295"><infon key="type">Gene</infon><location offset="21704" length="3"></location><text>GST</text></annotation><annotation id="Team286_A296"><infon key="type">Gene</infon><location offset="21749" length="5"></location><text>BamHI</text></annotation><annotation id="Team286_A297"><infon key="type">Gene</infon><location offset="21755" length="4"></location><text>XhoI</text></annotation><annotation id="Team286_A298"><infon key="type">Gene</infon><location offset="21773" length="7"></location><text>pKR17HA</text></annotation><annotation id="Team286_A299"><infon key="type">Gene</infon><location offset="21786" length="5"></location><text>pRD56</text></annotation><annotation id="Team286_A300"><infon key="type">Gene</infon><location offset="21880" length="6"></location><text>pRH254</text></annotation><annotation id="Team286_A301"><infon key="type">Gene</infon><location offset="21900" length="3"></location><text>GST</text></annotation><annotation id="Team286_A302"><infon key="type">Gene</infon><location offset="21904" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A303"><infon key="type">Gene</infon><location offset="21910" length="2"></location><text>HA</text></annotation><annotation id="Team286_A304"><infon key="type">Gene</infon><location offset="21936" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A305"><infon key="type">Gene</infon><location offset="21965" length="5"></location><text>pGAL1</text></annotation></sentence><sentence><offset>21972</offset><text>pPE123 is the SEC23 gene expressed from pGAL1 in pRS315 (Gimeno et al., 1996).</text><annotation id="Team286_A306"><infon key="type">Gene</infon><location offset="21972" length="6"></location><text>pPE123</text></annotation><annotation id="Team286_A307"><infon key="type">Gene</infon><location offset="21987" length="10"></location><text>SEC23 gene</text></annotation><annotation id="Team286_A308"><infon key="type">Gene</infon><location offset="22013" length="5"></location><text>pGAL1</text></annotation><annotation id="Team286_A309"><infon key="type">Gene</infon><location offset="22022" length="6"></location><text>pRS315</text></annotation></sentence><sentence><offset>22053</offset><text>Binding interactions were tested from extracts of CKY473 transformed with pRH254 (GST-Lst1p-HA) and either pCD43 (vector) or pPE123 (Sec23p).</text><annotation id="Team286_A310"><infon key="type">Gene</infon><location offset="22103" length="6"></location><text>CKY473</text></annotation><annotation id="Team286_A311"><infon key="type">Gene</infon><location offset="22128" length="6"></location><text>pRH254</text></annotation><annotation id="Team286_A312"><infon key="type">Gene</infon><location offset="22136" length="3"></location><text>GST</text></annotation><annotation id="Team286_A313"><infon key="type">Gene</infon><location offset="22140" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A314"><infon key="type">Gene</infon><location offset="22179" length="6"></location><text>pPE123</text></annotation><annotation id="Team286_A315"><infon key="type">Gene</infon><location offset="22187" length="6"></location><text>Sec23p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>22202</offset><sentence><offset>22202</offset><text>Cells were grown to exponential phase in SMM with 2% raffinose, galactose was added to 2%, and incubation was continued for 2 h at 30 C to induce pGAL1 expression.</text><annotation id="Team286_A316"><infon key="type">Gene</infon><location offset="22349" length="5"></location><text>pGAL1</text></annotation></sentence><sentence><offset>22367</offset><text>5 x 108 cells were converted to spheroplasts as previously described (Espenshade et al., 1995) and then gently lysed using glass beads in IP buffer (20 mM Hepes-KOH, pH 6.8, 80 mM KOAc, 5 mM magnesium acetate, 0.02% Triton X-100) containing the protease inhibitor cocktail.</text></sentence><sentence><offset>22644</offset><text>The extract was diluted to 1 ml with IP buffer, and membranes were collected by centrifugation at 500 g for 20 min.</text></sentence><sentence><offset>22760</offset><text>This pellet was extracted with 1 ml of IP buffer and 600 mM NaCl for 10 min at 0 C to release membrane-bound protein complexes.</text></sentence><sentence><offset>22889</offset><text>After clarification by centrifugation at 90,000 g for 10 min, the extract was diluted threefold with IP buffer, and a 1-ml aliquot was removed and incubated at room temperature for 1 h with glutathione Sepharose 4B beads (Pharmacia Biotech, Inc.).</text></sentence><sentence><offset>23139</offset><text>The beads were washed twice with 200 mM NaCl, 20 mM Hepes-KOH, pH 6.8, 80 mM KOAc, 5 mM magnesium acetate, 0.02% Triton X-100, and once in IP buffer without Triton X-100.</text></sentence><sentence><offset>23311</offset><text>Proteins were released from glutathione Sepharose 4B beads by solubilization in ESB.</text><annotation id="Team286_A317"><infon key="type">Gene</infon><location offset="23340" length="24"></location><text>glutathione Sepharose 4B</text></annotation></sentence><sentence><offset>23397</offset><text>Samples of total lysate were prepared by adding 2x ESB to an equal amount of the diluted extract from the salt washed membranes.</text></sentence><sentence><offset>23528</offset><text>Samples were analyzed by immunoblots probed with anti-Sec23p antibody.</text><annotation id="Team286_A318"><infon key="type">Gene</infon><location offset="23578" length="20"></location><text>anti-Sec23p antibody</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>23604</offset><sentence><offset>23604</offset><text>For analysis of the membrane association of GST-Lst1p-HA and Sec23p, cells expressing GST-Lst1p-HA, Sec23p, or both GST-Lst1p-HA and Sec23p from pGAL1, were grown in 2% raffinose and then induced by the addition of 2% galactose as described above.</text><annotation id="Team286_A319"><infon key="type">Gene</infon><location offset="23648" length="3"></location><text>GST</text></annotation><annotation id="Team286_A320"><infon key="type">Gene</infon><location offset="23652" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A321"><infon key="type">Gene</infon><location offset="23658" length="2"></location><text>HA</text></annotation><annotation id="Team286_A322"><infon key="type">Gene</infon><location offset="23666" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A323"><infon key="type">Gene</infon><location offset="23691" length="3"></location><text>GST</text></annotation><annotation id="Team286_A324"><infon key="type">Gene</infon><location offset="23695" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A325"><infon key="type">Gene</infon><location offset="23701" length="2"></location><text>HA</text></annotation><annotation id="Team286_A326"><infon key="type">Gene</infon><location offset="23705" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A327"><infon key="type">Gene</infon><location offset="23721" length="3"></location><text>GST</text></annotation><annotation id="Team286_A328"><infon key="type">Gene</infon><location offset="23725" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A329"><infon key="type">Gene</infon><location offset="23731" length="2"></location><text>HA</text></annotation><annotation id="Team286_A330"><infon key="type">Gene</infon><location offset="23739" length="17"></location><text>Sec23p from pGAL1</text></annotation></sentence><sentence><offset>23855</offset><text>2 h after induction, 2 x 107 cells were collected by centrifugation and resuspended in 20 mul of cell lysis buffer (20 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl2) with protease inhibitor cocktail.</text></sentence><sentence><offset>24051</offset><text>Cells were lysed by vigorous agitation with glass beads and an additional 500 mul of lysis buffer was added.</text></sentence><sentence><offset>24161</offset><text>The lysate was cleared of unlysed cells and large cell debris by centrifugation at 300 g for 3 min.</text></sentence><sentence><offset>24262</offset><text>50 mul of the supernatant was reserved for a total extract sample and the remainder was centrifuged to pellet ER membranes at 10,000 g for 30 min at 4 C in a microcentrifuge.</text></sentence><sentence><offset>24440</offset><text>An equal number of cell equivalents of total extract, membrane-pellet, and supernatant fractions was solubilized in ESB and analyzed by immunoblotting.</text></sentence><sentence><offset>24593</offset><text>The cytosolic protein Gdh2p was found only in the soluble fractions, demonstrating cell lysis was complete (data not shown).</text><annotation id="Team286_A331"><infon key="type">Gene</infon><location offset="24597" length="23"></location><text>cytosolic protein Gdh2p</text></annotation></sentence></passage><passage><infon key="type">title_1</infon><offset>24726</offset><sentence><offset>24726</offset><text>Results</text></sentence></passage><passage><infon key="type">title_2</infon><offset>24734</offset><sentence><offset>24734</offset><text>Mutations Synthetically Lethal with sec13-1</text><annotation id="Team286_A332"><infon key="type">Gene</infon><location offset="24770" length="7"></location><text>sec13-1</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>24778</offset><sentence><offset>24778</offset><text>To find new genes required for the budding of COPII vesicles, we screened for mutations that displayed synthetic lethality with the COPII mutation sec13-1 using a plasmid sectoring assay (Roberg et al., 1997b).</text><annotation id="Team286_A333"><infon key="type">Gene</infon><location offset="24911" length="22"></location><text>COPII mutation sec13-1</text></annotation></sentence><sentence><offset>24991</offset><text>Strain CKY423 has the chromosomal mutations ade2 ade3 sec13-1 and harbors the plasmid pKR4, which carries wild-type copies of SEC13 and ADE3.</text><annotation id="Team286_A334"><infon key="type">Gene</infon><location offset="25036" length="17"></location><text>ade2 ade3 sec13-1</text></annotation><annotation id="Team286_A335"><infon key="type">Gene</infon><location offset="25120" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A336"><infon key="type">Gene</infon><location offset="25130" length="4"></location><text>ADE3</text></annotation></sentence><sentence><offset>25136</offset><text>This strain accumulates a red pigment because of the ade2 mutation, but the spontaneous loss of pKR4 during the growth of a colony gives white sectors of ade2 ade3 segregants.</text><annotation id="Team286_A337"><infon key="type">Gene</infon><location offset="25190" length="13"></location><text>ade2 mutation</text></annotation><annotation id="Team286_A338"><infon key="type">Gene</infon><location offset="25234" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A339"><infon key="type">Gene</infon><location offset="25293" length="20"></location><text>ade2 ade3 segregants</text></annotation></sentence><sentence><offset>25315</offset><text>In this strain, a mutation that is lethal with sec13-1 will produce a nonsectoring colony.</text><annotation id="Team286_A340"><infon key="type">Gene</infon><location offset="25362" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>25406</offset><text>Mutagenesis of CKY423 and the isogenic strain of opposite mating type, CKY424, yielded 139 nonsectoring mutants (Fig. 1).</text><annotation id="Team286_A341"><infon key="type">Gene</infon><location offset="25421" length="6"></location><text>CKY423</text></annotation><annotation id="Team286_A342"><infon key="type">Gene</infon><location offset="25478" length="6"></location><text>CKY424</text></annotation></sentence><sentence><offset>25530</offset><text>These strains were then tested for restored ability to sector after transformation with pKR1, which carries wild-type SEC13, but lacks the ADE3 gene.</text><annotation id="Team286_A343"><infon key="type">Gene</infon><location offset="25619" length="4"></location><text>pKR1</text></annotation><annotation id="Team286_A344"><infon key="type">Gene</infon><location offset="25671" length="9"></location><text>ADE3 gene</text></annotation></sentence><sentence><offset>25682</offset><text>By this test, 57 of the mutants had synthetic-lethal mutations that could be rescued by wild-type SEC13.</text></sentence><sentence><offset>25789</offset><text>In backcrosses, 52 mutants gave a segregation pattern indicating that the trait was due to a single nuclear mutation (Fig. 1).</text><annotation id="Team286_A345"><infon key="type">Gene</infon><location offset="25908" length="6"></location><text>Fig. 1</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>25918</offset><sentence><offset>25918</offset><text>Matings between mutants identified 11 complementation groups using colony sectoring of the diploid as the criterion for allelic complementation.</text></sentence><sentence><offset>26063</offset><text>These complementation groups were designated LST (Table II).</text></sentence><sentence><offset>26124</offset><text>One of the complementation groups was shown to comprise recessive lethal mutations in the SEC13 gene itself (Roberg et al., 1997b).</text><annotation id="Team286_A346"><infon key="type">Gene</infon><location offset="26216" length="10"></location><text>SEC13 gene</text></annotation></sentence><sentence><offset>26259</offset><text>Tests for rescue of the nonsectoring phenotype by plasmids carrying known sec genes showed that LST10 was SEC16 (Roberg et al., 1997b).</text><annotation id="Team286_A347"><infon key="type">Gene</infon><location offset="26356" length="5"></location><text>LST10</text></annotation><annotation id="Team286_A348"><infon key="type">Gene</infon><location offset="26367" length="5"></location><text>SEC16</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>26398</offset><sentence><offset>26398</offset><text>Synthetic Interactions of lst Mutations</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>26438</offset><sentence><offset>26438</offset><text>To perform further genetic tests on the lst mutations, the lst sec13-1 double mutants were converted to lst single mutants by integration of a wild-type copy of SEC13 at the sec13-1 locus (Materials and Methods).</text><annotation id="Team286_A349"><infon key="type">Gene</infon><location offset="26502" length="22"></location><text>sec13-1 double mutants</text></annotation><annotation id="Team286_A350"><infon key="type">Gene</infon><location offset="26600" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A351"><infon key="type">Gene</infon><location offset="26614" length="13"></location><text>sec13-1 locus</text></annotation></sentence><sentence><offset>26653</offset><text>Representative lst single mutants were then crossed to sec16, sec23, and sec31 mutants.</text><annotation id="Team286_A352"><infon key="type">Gene</infon><location offset="26709" length="5"></location><text>sec16</text></annotation><annotation id="Team286_A353"><infon key="type">Gene</infon><location offset="26716" length="5"></location><text>sec23</text></annotation><annotation id="Team286_A354"><infon key="type">Gene</infon><location offset="26727" length="14"></location><text>sec31 mutants</text></annotation></sentence><sentence><offset>26743</offset><text>For mutations in LST2, LST3, LST4, LST5, LST7, and LST8, only crosses to sec13-1 gave a segregation pattern indicative of a synthetic-lethal interaction (Table III).</text><annotation id="Team286_A355"><infon key="type">Gene</infon><location offset="26760" length="4"></location><text>LST2</text></annotation><annotation id="Team286_A356"><infon key="type">Gene</infon><location offset="26766" length="4"></location><text>LST3</text></annotation><annotation id="Team286_A357"><infon key="type">Gene</infon><location offset="26772" length="4"></location><text>LST4</text></annotation><annotation id="Team286_A358"><infon key="type">Gene</infon><location offset="26778" length="4"></location><text>LST5</text></annotation><annotation id="Team286_A359"><infon key="type">Gene</infon><location offset="26785" length="4"></location><text>LST7</text></annotation><annotation id="Team286_A360"><infon key="type">Gene</infon><location offset="26795" length="4"></location><text>LST8</text></annotation><annotation id="Team286_A361"><infon key="type">Gene</infon><location offset="26817" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>26911</offset><text>We have subsequently shown that these LST genes relate to a function of SEC13 in the sorting of amino acid permeases in the late secretory pathway, and analysis of these genes is described elsewhere (Roberg et al., 1997a,b).</text><annotation id="Team286_A362"><infon key="type">Gene</infon><location offset="26949" length="10"></location><text>LST genes</text></annotation><annotation id="Team286_A363"><infon key="type">Gene</infon><location offset="26984" length="5"></location><text>SEC13</text></annotation></sentence><sentence><offset>27140</offset><text>Mutations in LST1 were inviable when combined with sec16, sec23, and sec31 mutations, and mutations in LST6 were inviable with sec16 and sec31 (Table III).</text><annotation id="Team286_A364"><infon key="type">Gene</infon><location offset="27153" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A365"><infon key="type">Gene</infon><location offset="27191" length="5"></location><text>sec16</text></annotation><annotation id="Team286_A366"><infon key="type">Gene</infon><location offset="27198" length="5"></location><text>sec23</text></annotation><annotation id="Team286_A367"><infon key="type">Gene</infon><location offset="27209" length="5"></location><text>sec31</text></annotation><annotation id="Team286_A368"><infon key="type">Gene</infon><location offset="27243" length="4"></location><text>LST6</text></annotation><annotation id="Team286_A369"><infon key="type">Gene</infon><location offset="27267" length="5"></location><text>sec16</text></annotation></sentence><sentence><offset>27297</offset><text>Importantly, mutations in LST1 and LST6 did not show synthetic lethality in parallel crosses to mutations in SEC17 or SEC18, genes required for fusion of COPII vesicles.</text><annotation id="Team286_A370"><infon key="type">Gene</infon><location offset="27323" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A371"><infon key="type">Gene</infon><location offset="27332" length="4"></location><text>LST6</text></annotation><annotation id="Team286_A372"><infon key="type">Gene</infon><location offset="27408" length="5"></location><text>SEC17</text></annotation><annotation id="Team286_A373"><infon key="type">Gene</infon><location offset="27417" length="5"></location><text>SEC18</text></annotation></sentence><sentence><offset>27469</offset><text>Given that synthetic-lethal interactions usually occur between mutations in genes involved in the same step of the secretory pathway, the tests for genetic interactions indicated that LST1, and probably also LST6, participate in vesicle budding from the ER.</text><annotation id="Team286_A374"><infon key="type">Gene</infon><location offset="27655" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A375"><infon key="type">Gene</infon><location offset="27679" length="4"></location><text>LST6</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>27731</offset><sentence><offset>27731</offset><text>Lst1p Is Homologous to Sec24p</text><annotation id="Team286_A376"><infon key="type">Gene</infon><location offset="27731" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A377"><infon key="type">Gene</infon><location offset="27754" length="6"></location><text>Sec24p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>27761</offset><sentence><offset>27761</offset><text>The LST1 gene was isolated by its ability to restore sectoring to CKY426 (MATa sec13-1 ade2 ade3 leu2 ura3 [pKR4]), a strain that forms solid red, nonsectoring colonies because of the presence of the lst1-1 mutation.</text><annotation id="Team286_A378"><infon key="type">Gene</infon><location offset="27765" length="9"></location><text>LST1 gene</text></annotation><annotation id="Team286_A379"><infon key="type">Gene</infon><location offset="27827" length="6"></location><text>CKY426</text></annotation><annotation id="Team286_A380"><infon key="type">Gene</infon><location offset="27835" length="17"></location><text>MATa sec13-1 ade2</text></annotation><annotation id="Team286_A381"><infon key="type">Gene</infon><location offset="27853" length="14"></location><text>ade3 leu2 ura3</text></annotation><annotation id="Team286_A382"><infon key="type">Gene</infon><location offset="27870" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A383"><infon key="type">Gene</infon><location offset="27962" length="6"></location><text>lst1-1</text></annotation></sentence><sentence><offset>27980</offset><text>CKY426 was transformed with yeast genomic libraries.</text><annotation id="Team286_A384"><infon key="type">Gene</infon><location offset="27980" length="6"></location><text>CKY426</text></annotation></sentence><sentence><offset>28033</offset><text>34 colonies that regained the ability to form white sectors were identified among 97,000 Ura+ transformants.</text></sentence><sentence><offset>28144</offset><text>We expected this screen to yield plasmids carrying either SEC13 or LST1.</text><annotation id="Team286_A385"><infon key="type">Gene</infon><location offset="28202" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A386"><infon key="type">Gene</infon><location offset="28212" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>28218</offset><text>About half of the complementing plasmids were shown to carry SEC13 by restriction site mapping and by the ability to complement the temperature sensitivity of sec13-1.</text><annotation id="Team286_A387"><infon key="type">Gene</infon><location offset="28280" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A388"><infon key="type">Gene</infon><location offset="28380" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>28389</offset><text>The restriction maps of the remaining rescuing plasmids show that they represent two unrelated chromosomal regions.</text></sentence><sentence><offset>28506</offset><text>The clones p21-31 and p77-2 were selected as representatives of each region.</text><annotation id="Team286_A389"><infon key="type">Gene</infon><location offset="28517" length="6"></location><text>p21-31</text></annotation><annotation id="Team286_A390"><infon key="type">Gene</infon><location offset="28528" length="5"></location><text>p77-2</text></annotation></sentence><sentence><offset>28584</offset><text>The genomic sequence from p77-2 (a clone in the plambdaYES vector; Elledge et al., 1991) was inserted as an XhoI fragment into the integrating vector pRS306 to produce pKR20.</text><annotation id="Team286_A391"><infon key="type">Gene</infon><location offset="28611" length="5"></location><text>p77-2</text></annotation><annotation id="Team286_A392"><infon key="type">Gene</infon><location offset="28694" length="4"></location><text>XhoI</text></annotation><annotation id="Team286_A393"><infon key="type">Gene</infon><location offset="28754" length="5"></location><text>pKR20</text></annotation></sentence><sentence><offset>28761</offset><text>For chromosomal integration, pKR20 was linearized by digestion with HpaI and transformed into CUY564 (MATalpha ade2 ade3 leu2 ura3).</text><annotation id="Team286_A394"><infon key="type">Gene</infon><location offset="28791" length="5"></location><text>pKR20</text></annotation><annotation id="Team286_A395"><infon key="type">Gene</infon><location offset="28830" length="4"></location><text>HpaI</text></annotation><annotation id="Team286_A396"><infon key="type">Gene</infon><location offset="28857" length="6"></location><text>CUY564</text></annotation><annotation id="Team286_A397"><infon key="type">Gene</infon><location offset="28865" length="29"></location><text>MATalpha ade2 ade3 leu2 ura3</text></annotation></sentence><sentence><offset>28897</offset><text>The resulting strain was crossed to the lst1-1 mutant CKY426 (MATa lst1-1 sec13-1 ade2 ade3 leu2 ura3 [pKR4]).</text><annotation id="Team286_A398"><infon key="type">Gene</infon><location offset="28937" length="21"></location><text>lst1-1 mutant CKY426</text></annotation><annotation id="Team286_A399"><infon key="type">Gene</infon><location offset="28960" length="4"></location><text>MATa</text></annotation><annotation id="Team286_A400"><infon key="type">Gene</infon><location offset="28965" length="19"></location><text>lst1-1 sec13-1 ade2</text></annotation><annotation id="Team286_A401"><infon key="type">Gene</infon><location offset="28985" length="14"></location><text>ade3 leu2 ura3</text></annotation><annotation id="Team286_A402"><infon key="type">Gene</infon><location offset="29002" length="4"></location><text>pKR4</text></annotation></sentence><sentence><offset>29010</offset><text>After sporulation and dissection, the integrated pKR20 was found to be completely linked to the LST1 locus: sectoring segregated 2:2 and all sectored colonies were Ura+, whereas all nonsectored colonies were Ura-.</text><annotation id="Team286_A403"><infon key="type">Gene</infon><location offset="29060" length="5"></location><text>pKR20</text></annotation><annotation id="Team286_A404"><infon key="type">Gene</infon><location offset="29107" length="10"></location><text>LST1 locus</text></annotation><annotation id="Team286_A405"><infon key="type">Gene</infon><location offset="29176" length="4"></location><text>Ura+</text></annotation><annotation id="Team286_A406"><infon key="type">Gene</infon><location offset="29220" length="3"></location><text>Ura</text></annotation></sentence><sentence><offset>29227</offset><text>Thus, p77-2 carries the LST1 gene.</text><annotation id="Team286_A407"><infon key="type">Gene</infon><location offset="29233" length="5"></location><text>p77-2</text></annotation><annotation id="Team286_A408"><infon key="type">Gene</infon><location offset="29251" length="9"></location><text>LST1 gene</text></annotation></sentence><sentence><offset>29262</offset><text>In parallel, the genomic sequence from p21-31 (a clone in the pCT3 vector; Thompson et al., 1993) was inserted as an EcoRI/HindIII fragment into pRS306 to produce pKR7.</text><annotation id="Team286_A409"><infon key="type">Gene</infon><location offset="29301" length="6"></location><text>p21-31</text></annotation><annotation id="Team286_A410"><infon key="type">Gene</infon><location offset="29380" length="5"></location><text>EcoRI</text></annotation><annotation id="Team286_A411"><infon key="type">Gene</infon><location offset="29386" length="7"></location><text>HindIII</text></annotation><annotation id="Team286_A412"><infon key="type">Gene</infon><location offset="29409" length="6"></location><text>pRS306</text></annotation><annotation id="Team286_A413"><infon key="type">Gene</infon><location offset="29427" length="4"></location><text>pKR7</text></annotation></sentence><sentence><offset>29433</offset><text>pKR7 was integrated at its chromosomal locus after linearization with MscI and was then checked for linkage to lst1-1.</text><annotation id="Team286_A414"><infon key="type">Gene</infon><location offset="29433" length="4"></location><text>pKR7</text></annotation><annotation id="Team286_A415"><infon key="type">Gene</infon><location offset="29504" length="4"></location><text>MscI</text></annotation><annotation id="Team286_A416"><infon key="type">Gene</infon><location offset="29545" length="6"></location><text>lst1-1</text></annotation></sentence><sentence><offset>29553</offset><text>Tetrad analysis showed that pKR7 was not linked to LST1 and we concluded that pKR7 carries an unlinked suppressor gene.</text><annotation id="Team286_A417"><infon key="type">Gene</infon><location offset="29582" length="4"></location><text>pKR7</text></annotation><annotation id="Team286_A418"><infon key="type">Gene</infon><location offset="29605" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A419"><infon key="type">Gene</infon><location offset="29633" length="4"></location><text>pKR7</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>29670</offset><sentence><offset>29670</offset><text>The 3.5-kb insert of p77-2 was inserted into the XhoI site of the centromeric vector pRS316 to construct pKR17.</text><annotation id="Team286_A420"><infon key="type">Gene</infon><location offset="29691" length="5"></location><text>p77-2</text></annotation><annotation id="Team286_A421"><infon key="type">Gene</infon><location offset="29719" length="10"></location><text>XhoI site</text></annotation></sentence><sentence><offset>29784</offset><text>The base sequence of this insert was determined and found to contain a single open reading frame encoding a protein of 929 amino acids.</text></sentence><sentence><offset>29922</offset><text>This sequence corresponds to the open reading frame YHR098c located on chromosome VIII (Saccharomyces Genome Database, Cherry et al., 1997).</text></sentence><sentence><offset>30066</offset><text>The predicted amino acid sequence of LST1 shows significant similarity to SEC24 (YIL109C).</text><annotation id="Team286_A422"><infon key="type">Gene</infon><location offset="30103" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A423"><infon key="type">Gene</infon><location offset="30141" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A424"><infon key="type">Gene</infon><location offset="30148" length="7"></location><text>YIL109C</text></annotation></sentence><sentence><offset>30158</offset><text>The two proteins share 23% sequence identity that extends over most of their length (Fig. 2), suggesting that Lst1p may have a function similar to that of Sec24p as a subunit of the COPII vesicle coat.</text><annotation id="Team286_A425"><infon key="type">Gene</infon><location offset="30269" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A426"><infon key="type">Gene</infon><location offset="30315" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A427"><infon key="type">Gene</infon><location offset="30343" length="18"></location><text>COPII vesicle coat</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>30364</offset><sentence><offset>30364</offset><text>Phenotypes of lst1Delta</text><annotation id="Team286_A428"><infon key="type">Gene</infon><location offset="30378" length="9"></location><text>lst1Delta</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>30385</offset><sentence><offset>30385</offset><text>One copy of the LST1 gene in the wild-type diploid strain CKY348 was disrupted to generate a lst1Delta::LEU2/LST1 heterozygote.</text><annotation id="Team286_A429"><infon key="type">Gene</infon><location offset="30401" length="9"></location><text>LST1 gene</text></annotation><annotation id="Team286_A430"><infon key="type">Gene</infon><location offset="30490" length="4"></location><text>LEU2</text></annotation><annotation id="Team286_A431"><infon key="type">Gene</infon><location offset="30495" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>30515</offset><text>Sporulation and dissection of this diploid gave &gt;95% spore viability on YPD medium and the LEU2 marker segregated 2:2, showing that LST1 is not essential for growth.</text><annotation id="Team286_A432"><infon key="type">Gene</infon><location offset="30608" length="4"></location><text>LEU2</text></annotation><annotation id="Team286_A433"><infon key="type">Gene</infon><location offset="30649" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>30683</offset><text>A lst1Delta::LEU2 mutant spore clone was crossed to sec mutants to test for synthetic lethality.</text><annotation id="Team286_A434"><infon key="type">Gene</infon><location offset="30696" length="23"></location><text>LEU2 mutant spore clone</text></annotation></sentence><sentence><offset>30781</offset><text>In these crosses, both the temperature sensitivity of the sec mutation and the lst1Delta allele marked by LEU2 could be followed independently.</text><annotation id="Team286_A435"><infon key="type">Gene</infon><location offset="30862" length="16"></location><text>lst1Delta allele</text></annotation><annotation id="Team286_A436"><infon key="type">Gene</infon><location offset="30889" length="4"></location><text>LEU2</text></annotation></sentence><sentence><offset>30928</offset><text>In crosses of lst1Delta to sec12, sec13, sec16, sec23, sec24, or sec31 mutants, inviability segregated as a two-gene trait (segregation patterns for dead:viable spore clones were 2:2, 1:3, and 0:4).</text><annotation id="Team286_A437"><infon key="type">Gene</infon><location offset="30942" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A438"><infon key="type">Gene</infon><location offset="30955" length="5"></location><text>sec12</text></annotation><annotation id="Team286_A439"><infon key="type">Gene</infon><location offset="30962" length="5"></location><text>sec13</text></annotation><annotation id="Team286_A440"><infon key="type">Gene</infon><location offset="30970" length="5"></location><text>sec16</text></annotation><annotation id="Team286_A441"><infon key="type">Gene</infon><location offset="30977" length="5"></location><text>sec23</text></annotation><annotation id="Team286_A442"><infon key="type">Gene</infon><location offset="30984" length="5"></location><text>sec24</text></annotation><annotation id="Team286_A443"><infon key="type">Gene</infon><location offset="30994" length="13"></location><text>sec31 mutants</text></annotation></sentence><sentence><offset>31130</offset><text>Tests of the genotype of the surviving sister spore clones showed that the inviable spores in these crosses were always lst1Delta sec double mutants.</text><annotation id="Team286_A444"><infon key="type">Gene</infon><location offset="31251" length="29"></location><text>lst1Delta sec double mutants</text></annotation></sentence><sentence><offset>31282</offset><text>Crosses between lst1Delta and sec17 or sec18 produced viable double mutants.</text><annotation id="Team286_A445"><infon key="type">Gene</infon><location offset="31298" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A446"><infon key="type">Gene</infon><location offset="31312" length="5"></location><text>sec17</text></annotation><annotation id="Team286_A447"><infon key="type">Gene</infon><location offset="31321" length="5"></location><text>sec18</text></annotation></sentence><sentence><offset>31359</offset><text>These findings confirmed and extended our earlier tests for synthetic lethality with lst1-1, and demonstrated that lst1Delta was synthetically lethal with all the known genes required for COPII vesicle formation, but not with genes required for vesicle fusion.</text><annotation id="Team286_A448"><infon key="type">Gene</infon><location offset="31446" length="6"></location><text>lst1-1</text></annotation><annotation id="Team286_A449"><infon key="type">Gene</infon><location offset="31476" length="9"></location><text>lst1Delta</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>31602</offset><sentence><offset>31602</offset><text>We evaluated the growth of lst1Delta::LEU2 mutants under a variety of conditions.</text><annotation id="Team286_A450"><infon key="type">Gene</infon><location offset="31629" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A451"><infon key="type">Gene</infon><location offset="31640" length="12"></location><text>LEU2 mutants</text></annotation></sentence><sentence><offset>31685</offset><text>On YPD, the lst1Delta::LEU2 strain grew, as well as an isogenic wild-type strain at temperatures ranging from 14 to 37 C.</text><annotation id="Team286_A452"><infon key="type">Gene</infon><location offset="31697" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A453"><infon key="type">Gene</infon><location offset="31708" length="4"></location><text>LEU2</text></annotation></sentence><sentence><offset>31808</offset><text>However, on SMM the lst1Delta::LEU2 strain grew poorly at temperatures above 30 C.</text><annotation id="Team286_A454"><infon key="type">Gene</infon><location offset="31840" length="4"></location><text>LEU2</text></annotation></sentence><sentence><offset>31893</offset><text>Since YPD (pH 6.5) and SMM (pH 3.8) differed markedly in pH, we suspected that lst1Delta mutants may be particularly sensitive to an acidic environment, and we tested the effect of pH on the growth of lst1Delta mutants.</text><annotation id="Team286_A455"><infon key="type">Gene</infon><location offset="31973" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A456"><infon key="type">Gene</infon><location offset="32097" length="17"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>32116</offset><text>Although lst1Delta mutants grew as well as wild-type on YPD at all temperatures, when YPD was brought to pH 3.8, lst1Delta mutants grew much more slowly than wild-type at 37 C (Fig. 3 A).</text><annotation id="Team286_A457"><infon key="type">Gene</infon><location offset="32125" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A458"><infon key="type">Gene</infon><location offset="32222" length="6"></location><text>pH 3.8</text></annotation><annotation id="Team286_A459"><infon key="type">Gene</infon><location offset="32230" length="18"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>32307</offset><text>Conversely, on SMM buffered to pH 6.5, lst1Delta and wild-type grew even at 37 C (data not shown).</text></sentence><sentence><offset>32407</offset><text>These results demonstrated that at high temperature, growth of the lst1Delta mutant was sensitive to acidic conditions.</text><annotation id="Team286_A460"><infon key="type">Gene</infon><location offset="32476" length="16"></location><text>lst1Delta mutant</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>32507</offset><sentence><offset>32507</offset><text>Having identified conditions where LST1 was needed for growth, we investigated whether overexpression of SEC24 could supply the function lost in lst1Delta.</text><annotation id="Team286_A461"><infon key="type">Gene</infon><location offset="32542" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A462"><infon key="type">Gene</infon><location offset="32614" length="5"></location><text>SEC24</text></annotation></sentence><sentence><offset>32665</offset><text>Some restoration of function was indicated by the ability of an lst1Delta mutant to grow on acidic medium when provided with extra copies of SEC24 on either centromeric or 2mu plasmids (Fig. 3 B).</text><annotation id="Team286_A463"><infon key="type">Gene</infon><location offset="32729" length="17"></location><text>lst1Delta mutant</text></annotation><annotation id="Team286_A464"><infon key="type">Gene</infon><location offset="32807" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A465"><infon key="type">Gene</infon><location offset="32853" length="8"></location><text>Fig. 3 B</text></annotation></sentence><sentence><offset>32864</offset><text>These findings imply some functional overlap between LST1 and SEC24.</text><annotation id="Team286_A466"><infon key="type">Gene</infon><location offset="32918" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A467"><infon key="type">Gene</infon><location offset="32927" length="5"></location><text>SEC24</text></annotation></sentence><sentence><offset>32934</offset><text>In parallel tests for suppression, we found that the genes SEC12, SEC13, SEC31, or SEC23, when expressed from 2mu plasmids, could not restore the ability of an lst1Delta mutant to grow on acidic medium.</text><annotation id="Team286_A468"><infon key="type">Gene</infon><location offset="32993" length="5"></location><text>SEC12</text></annotation><annotation id="Team286_A469"><infon key="type">Gene</infon><location offset="33000" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A470"><infon key="type">Gene</infon><location offset="33007" length="5"></location><text>SEC31</text></annotation><annotation id="Team286_A471"><infon key="type">Gene</infon><location offset="33018" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A472"><infon key="type">Gene</infon><location offset="33095" length="16"></location><text>lst1Delta mutant</text></annotation></sentence><sentence><offset>33138</offset><text>We found that the lst1Delta mutation caused a selective defect in the trafficking of Pma1p from the ER, and we also examined the ability of overexpressed SEC24 to suppress this phenotype caused by the lst1Delta mutation.</text><annotation id="Team286_A473"><infon key="type">Gene</infon><location offset="33156" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A474"><infon key="type">Gene</infon><location offset="33224" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A475"><infon key="type">Gene</infon><location offset="33294" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A476"><infon key="type">Gene</infon><location offset="33341" length="9"></location><text>lst1Delta</text></annotation></sentence><sentence><offset>33361</offset><text>By immunofluorescence microscopy, the proper localization of Pma1p to the cell surface was restored in an lst1Delta strain that also carried SEC24 on a 2mu plasmid (see Fig. 5).</text><annotation id="Team286_A477"><infon key="type">Gene</infon><location offset="33423" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A478"><infon key="type">Gene</infon><location offset="33468" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A479"><infon key="type">Gene</infon><location offset="33504" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A480"><infon key="type">Gene</infon><location offset="33532" length="6"></location><text>Fig. 5</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>33524</offset><sentence><offset>33524</offset><text>In an attempt to test the effect of overexpression of LST1, we found that LST1 on a 2mu plasmid severely impaired growth of wild-type yeast cells.</text><annotation id="Team286_A481"><infon key="type">Gene</infon><location offset="33579" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A482"><infon key="type">Gene</infon><location offset="33599" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>33672</offset><text>To examine the response of cells to different doses of Lst1p, we designed a way to express different levels of Lst1p according to the amount of galactose in the growth medium.</text><annotation id="Team286_A483"><infon key="type">Gene</infon><location offset="33727" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A484"><infon key="type">Gene</infon><location offset="33784" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>33850</offset><text>A wild-type strain (CKY473) carrying a plasmid that expressed LST1 from pGAL1 (pKR35) was spread on an SMM plate with 2% raffinose, a carbon source that allows yeast growth without repression of the GAL1 promoter.</text><annotation id="Team286_A485"><infon key="type">Gene</infon><location offset="33913" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A486"><infon key="type">Gene</infon><location offset="33924" length="5"></location><text>pGAL1</text></annotation><annotation id="Team286_A487"><infon key="type">Gene</infon><location offset="33931" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A488"><infon key="type">Gene</infon><location offset="34053" length="13"></location><text>GAL1 promoter</text></annotation></sentence><sentence><offset>34068</offset><text>When these cells are exposed to a gradient of galactose concentrations, from 3 mg of galactose in a filter disk on top of the lawn, growth was inhibited in a halo 1.5 cm beyond the edge of the filter (Fig. 3 C).</text></sentence><sentence><offset>34284</offset><text>A strain that did not contain pKR35 grew uniformly up to the edge of the filter, showing that the galactose itself was not inhibitory.</text><annotation id="Team286_A489"><infon key="type">Gene</infon><location offset="34314" length="5"></location><text>pKR35</text></annotation></sentence><sentence><offset>34421</offset><text>Given the similarity of Lst1p to Sec24p, we asked whether the overexpression of SEC24 could compensate for overexpression of LST1.</text><annotation id="Team286_A490"><infon key="type">Gene</infon><location offset="34446" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A491"><infon key="type">Gene</infon><location offset="34455" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A492"><infon key="type">Gene</infon><location offset="34503" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A493"><infon key="type">Gene</infon><location offset="34548" length="4"></location><text>LST1</text></annotation></sentence><sentence><offset>34555</offset><text>Cells carrying both the pGAL1-LST1 plasmid (pKR35) and the SEC24 gene on a 2mu plasmid (pKR41) were tested in an identical halo assay, and were found to be resistant to the effect of galactose (Fig. 3 C).</text><annotation id="Team286_A494"><infon key="type">Gene</infon><location offset="34579" length="18"></location><text>pGAL1-LST1 plasmid</text></annotation><annotation id="Team286_A495"><infon key="type">Gene</infon><location offset="34599" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A496"><infon key="type">Gene</infon><location offset="34615" length="10"></location><text>SEC24 gene</text></annotation><annotation id="Team286_A497"><infon key="type">Gene</infon><location offset="34644" length="5"></location><text>pKR41</text></annotation></sentence><sentence><offset>34763</offset><text>Suppression by SEC24 appeared to be specific, since parallel tests of 2mu plasmids carrying SEC12, SEC13, SEC31, or SEC23 failed to show suppression.</text><annotation id="Team286_A498"><infon key="type">Gene</infon><location offset="34778" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A499"><infon key="type">Gene</infon><location offset="34857" length="5"></location><text>SEC12</text></annotation><annotation id="Team286_A500"><infon key="type">Gene</infon><location offset="34864" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A501"><infon key="type">Gene</infon><location offset="34871" length="5"></location><text>SEC31</text></annotation><annotation id="Team286_A502"><infon key="type">Gene</infon><location offset="34881" length="5"></location><text>SEC23</text></annotation></sentence><sentence><offset>34916</offset><text>It is worth noting that SEC23 expressed from a 2mu plasmid significantly slows the growth of our yeast strains.</text><annotation id="Team286_A503"><infon key="type">Gene</infon><location offset="34940" length="5"></location><text>SEC23</text></annotation></sentence><sentence><offset>35030</offset><text>Any suppression afforded by overexpression of SEC23 might be counteracted by this inherent toxicity of SEC23.</text><annotation id="Team286_A504"><infon key="type">Gene</infon><location offset="35077" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A505"><infon key="type">Gene</infon><location offset="35135" length="5"></location><text>SEC23</text></annotation></sentence><sentence><offset>35142</offset><text>A simple conclusion that can be drawn from these overexpression studies is that too great of a stoichiometric excess of Lst1p over Sec24p is lethal.</text><annotation id="Team286_A506"><infon key="type">Gene</infon><location offset="35264" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>35293</offset><text>This observation can be explained if Lst1p and Sec24p compete with one another in the assembly of vesicle coat complexes and that excess Lst1p causes sequestration of vesicle components into complexes that fail to satisfy some essential function of COPII.</text><annotation id="Team286_A507"><infon key="type">Gene</infon><location offset="35331" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A508"><infon key="type">Gene</infon><location offset="35341" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A509"><infon key="type">Gene</infon><location offset="35392" length="22"></location><text>vesicle coat complexes</text></annotation><annotation id="Team286_A510"><infon key="type">Gene</infon><location offset="35432" length="5"></location><text>Lst1p</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>35555</offset><sentence><offset>35555</offset><text>lst1Delta Diminishes the Activity of the Plasma Membrane Proton-ATPase</text><annotation id="Team286_A511"><infon key="type">Gene</infon><location offset="35555" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A512"><infon key="type">Gene</infon><location offset="35596" length="29"></location><text>Plasma Membrane Proton-ATPase</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>35623</offset><sentence><offset>35623</offset><text>The sensitivity of lst1Delta mutants to low pH suggested the involvement of Pma1p, which has been shown to be the limiting cell component for growth on acidic medium (McCusker et al., 1987; Portillo and Serrano, 1989).</text><annotation id="Team286_A513"><infon key="type">Gene</infon><location offset="35642" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A514"><infon key="type">Gene</infon><location offset="35700" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>35844</offset><text>The dependence of Pma1p activity on LST1 was supported by the observation that lst1Delta mutants exhibited an unusual morphology characteristic of pma1 mutants.</text><annotation id="Team286_A515"><infon key="type">Gene</infon><location offset="35862" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A516"><infon key="type">Gene</infon><location offset="35880" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A517"><infon key="type">Gene</infon><location offset="35924" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A518"><infon key="type">Gene</infon><location offset="35992" length="12"></location><text>pma1 mutants</text></annotation></sentence><sentence><offset>36006</offset><text>When lst1Delta mutants were grown in low pH (SMM or YPD brought to pH 3.8) at 37 C, ~10% of the cells formed multibudded rosettes; in some cases, as many as 15 daughters radiated from a single large mother cell (Fig. 4 A).</text><annotation id="Team286_A519"><infon key="type">Gene</infon><location offset="36011" length="17"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>36231</offset><text>The unseparated daughter cells contained nuclei that could be stained with DAPI and the daughter cells could be separated from their mothers by micromanipulation, indicating they had completed cytokinesis.</text></sentence><sentence><offset>36441</offset><text>Cells depleted of Pma1p produce similar multibudded cells with attached daughters that had completed cytokinesis.</text><annotation id="Team286_A520"><infon key="type">Gene</infon><location offset="36459" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>36557</offset><text>In this case, multibudded rosettes are thought to form because a mother cell formed with sufficient Pma1p in the plasma membrane will continue to bud, whereas daughter cells formed after Pma1p transport is compromised will have insufficient Pma1p to form buds themselves (Cid et al., 1987).</text><annotation id="Team286_A521"><infon key="type">Gene</infon><location offset="36659" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A522"><infon key="type">Gene</infon><location offset="36747" length="15"></location><text>Pma1p transport</text></annotation><annotation id="Team286_A523"><infon key="type">Gene</infon><location offset="36802" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>36853</offset><text>The morphology of lst1Delta cells grown at relatively high pH (YPD or SMM buffered to pH 6.5) at 37 C appeared normal, with few cells having more than one attached daughter.</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>37023</offset><sentence><offset>37023</offset><text>As a more direct test of the effect of lst1Delta on the activity of Pma1p, we measured the capacity of mutant cells to pump protons into the external medium.</text><annotation id="Team286_A524"><infon key="type">Gene</infon><location offset="37062" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A525"><infon key="type">Gene</infon><location offset="37092" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>37183</offset><text>Wild-type and lst1Delta strains were cultured in YPD at 37 C, conditions under which both strains grow equally well.</text></sentence><sentence><offset>37301</offset><text>After starvation by prolonged incubation in water, the cells were placed in a weakly buffered medium and proton efflux on addition of glucose was measured as a drop in extracellular pH.</text></sentence><sentence><offset>37490</offset><text>For both wild-type and lst1Delta strains, addition of glucose produced a sharp decline in pH (after a 30-s lag), which began to level off after ~5 min (Fig. 4 B).</text><annotation id="Team286_A526"><infon key="type">Gene</infon><location offset="37644" length="8"></location><text>Fig. 4 B</text></annotation></sentence><sentence><offset>37655</offset><text>Although the responses of wild-type and lst1Delta cells were qualitatively similar, proton efflux from lst1Delta cells was compromised: in the first 5 min after addition of glucose the rate of change in pH produced by the lst1Delta mutant was 65% of that of wild-type.</text></sentence><sentence><offset>37928</offset><text>These findings indicate that the lst1Delta mutant grown at 37 C has about half of the Pma1p activity as wild-type cells.</text><annotation id="Team286_A527"><infon key="type">Gene</infon><location offset="37961" length="16"></location><text>lst1Delta mutant</text></annotation><annotation id="Team286_A528"><infon key="type">Gene</infon><location offset="38015" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>38033</offset><sentence><offset>38033</offset><text>LST1 Is Required for Efficient Transport of Pma1p Out of the ER</text><annotation id="Team286_A529"><infon key="type">Gene</infon><location offset="38033" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A530"><infon key="type">Gene</infon><location offset="38077" length="9"></location><text>Pma1p Out</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>38097</offset><sentence><offset>38097</offset><text>To determine whether the reduced Pma1p activity in lst1Delta mutants was due to a defect in the transport of Pma1p to the cell surface, we compared the localization of Pma1p in wild-type and lst1Delta mutant cells by immunofluorescence microscopy.</text><annotation id="Team286_A531"><infon key="type">Gene</infon><location offset="38130" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A532"><infon key="type">Gene</infon><location offset="38148" length="18"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A533"><infon key="type">Gene</infon><location offset="38207" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A534"><infon key="type">Gene</infon><location offset="38267" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>38349</offset><text>Cells were grown at 30 C in YPD medium to avoid possible secondary effects due to the pH sensitivity of lst1Delta mutants.</text><annotation id="Team286_A535"><infon key="type">Gene</infon><location offset="38455" length="17"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>38474</offset><text>In lst1Delta cells, Pma1p was located primarily at the nuclear periphery and at the cellular rim, indicating that a large proportion of Pma1p remains in the ER (Fig. 5).</text><annotation id="Team286_A536"><infon key="type">Gene</infon><location offset="38494" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A537"><infon key="type">Gene</infon><location offset="38610" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>38645</offset><text>This pattern of localization differed markedly from the surface localization of Pma1p in wild-type cells incubated at 30 C (Fig. 5) or in lst1Delta cells incubated at 24 C (data not shown).</text><annotation id="Team286_A538"><infon key="type">Gene</infon><location offset="38726" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>38826</offset><sentence><offset>38826</offset><text>We also examined the subcellular distribution of Pma1p in lst1Delta cells by cell fractionation.</text><annotation id="Team286_A539"><infon key="type">Gene</infon><location offset="38875" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>38924</offset><text>Lysates from cells grown at 37 C for 3 h were fractionated on sucrose density gradients under conditions where the ER and plasma membrane are well separated on the basis of their buoyant density.</text></sentence><sentence><offset>39121</offset><text>Pma1p from wild-type cells was located in dense fractions of the gradient in a peak that was coincident with that of Gas1p, a GPI-linked plasma membrane protein (Nuoffer et al., 1991).</text><annotation id="Team286_A540"><infon key="type">Gene</infon><location offset="39121" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A541"><infon key="type">Gene</infon><location offset="39240" length="5"></location><text>Gas1p</text></annotation></sentence><sentence><offset>39309</offset><text>In contrast, &lt;35% of the total Pma1p from lst1Delta cells coincided with the plasma membrane marked by Gas1p protein and the majority of Pma1p was located in fractions containing the ER (Fig. 6).</text><annotation id="Team286_A542"><infon key="type">Gene</infon><location offset="39341" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A543"><infon key="type">Gene</infon><location offset="39413" length="13"></location><text>Gas1p protein</text></annotation><annotation id="Team286_A544"><infon key="type">Gene</infon><location offset="39448" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>39508</offset><text>Interestingly, the ER from lst1Delta mutants (marked by Sec61p) reproducibly resolved into two peaks of different density, suggesting that accumulation of Pma1p segregates ER membranes into subdomains of relatively high and low density.</text><annotation id="Team286_A545"><infon key="type">Gene</infon><location offset="39527" length="25"></location><text>ER from lst1Delta mutants</text></annotation><annotation id="Team286_A546"><infon key="type">Gene</infon><location offset="39565" length="6"></location><text>Sec61p</text></annotation><annotation id="Team286_A547"><infon key="type">Gene</infon><location offset="39665" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>39749</offset><text>Given that most of the Pma1p was located in the ER peak of higher density, it is possible that the density of the ER had been increased because of the accumulation of Pma1p.</text><annotation id="Team286_A548"><infon key="type">Gene</infon><location offset="39772" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A549"><infon key="type">Gene</infon><location offset="39919" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>39926</offset><text>A similar increase in density of a portion of the ER is caused when folding mutants of PMA1 are retained within the ER (Harris et al., 1994).</text><annotation id="Team286_A550"><infon key="type">Gene</infon><location offset="39977" length="2"></location><text>ER</text></annotation><annotation id="Team286_A551"><infon key="type">Gene</infon><location offset="40014" length="4"></location><text>PMA1</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>40063</offset><sentence><offset>40063</offset><text>The fact that transport of Pma1p, but not of Gas1p, was affected by deletion of LST1 suggested that LST1 may be specifically required for the export of Pma1p from the ER.</text><annotation id="Team286_A552"><infon key="type">Gene</infon><location offset="40090" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A553"><infon key="type">Gene</infon><location offset="40108" length="5"></location><text>Gas1p</text></annotation><annotation id="Team286_A554"><infon key="type">Gene</infon><location offset="40144" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A555"><infon key="type">Gene</infon><location offset="40164" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A556"><infon key="type">Gene</infon><location offset="40217" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>40237</offset><text>The absence of a general protein secretion defect in lst1Delta mutants was implied by the normal growth of lst1Delta mutants at 37 C in medium of pH 6.5 (the doubling time of both lst1Delta and wild-type was 1.75 h in YPD), indicating a normal rate of expansion of the plasma membrane.</text><annotation id="Team286_A557"><infon key="type">Gene</infon><location offset="40290" length="18"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A558"><infon key="type">Gene</infon><location offset="40345" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A559"><infon key="type">Gene</infon><location offset="40419" length="9"></location><text>lst1Delta</text></annotation></sentence><sentence><offset>40526</offset><text>As a specific test for the rate of ER to Golgi transport, pulse- chase experiments were performed to follow the rate of maturation of invertase from its core glycosylated ER form to the Golgi and secreted forms.</text><annotation id="Team286_A560"><infon key="type">Gene</infon><location offset="40662" length="9"></location><text>invertase</text></annotation></sentence><sentence><offset>40741</offset><text>No delay in invertase transport was observed in lst1Delta mutants that had been grown at 37 C for 3 h, conditions that caused the accumulation of Pma1p (Fig. 7).</text><annotation id="Team286_A561"><infon key="type">Gene</infon><location offset="40753" length="9"></location><text>invertase</text></annotation></sentence><sentence><offset>40904</offset><text>Similarly, no defect in the maturation of carboxypeptidase Y from the ER form to the Golgi and vacuolar forms of the enzyme could be detected (data not shown).</text><annotation id="Team286_A562"><infon key="type">Gene</infon><location offset="40946" length="18"></location><text>carboxypeptidase Y</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>41059</offset><sentence><offset>41059</offset><text>We also considered the possibility that transport of Pma1p may be particularly sensitive to any subtle defect in vesicle formation.</text><annotation id="Team286_A563"><infon key="type">Gene</infon><location offset="41113" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>41193</offset><text>We addressed this possibility by examining the localization of Pma1p in sec24-1 and sec31-2 mutant cells at the semipermissive temperature of 28 C.</text><annotation id="Team286_A564"><infon key="type">Gene</infon><location offset="41256" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A565"><infon key="type">Gene</infon><location offset="41265" length="7"></location><text>sec24-1</text></annotation><annotation id="Team286_A566"><infon key="type">Gene</infon><location offset="41277" length="7"></location><text>sec31-2</text></annotation></sentence><sentence><offset>41341</offset><text>Although the growth rate of both mutants was compromised at this temperature (doubling time on YPD: 2.9 h for sec24 and 2.4 h for sec31, as compared with 1.7 h for wild-type), no accumulation of Pma1p was detected in the perinuclear region of either mutant by immunofluorescence microscopy (data not shown).</text><annotation id="Team286_A567"><infon key="type">Gene</infon><location offset="41539" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>41653</offset><text>Thus, partial defects in COPII functions did not lead to the extensive accumulation of Pma1p in the ER that was observed for lst1Delta mutants.</text><annotation id="Team286_A568"><infon key="type">Gene</infon><location offset="41742" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A569"><infon key="type">Gene</infon><location offset="41780" length="17"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>41800</offset><text>Taken together, comparisons between the lst1Delta mutation and COPII gene mutations indicate that the lst1Delta mutation is unusual in its ability to inhibit Pma1p exit from the ER without interfering with the transport of other cargo proteins.</text><annotation id="Team286_A570"><infon key="type">Gene</infon><location offset="41864" length="10"></location><text>COPII gene</text></annotation><annotation id="Team286_A571"><infon key="type">Gene</infon><location offset="41903" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A572"><infon key="type">Gene</infon><location offset="41959" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>42040</offset><sentence><offset>42040</offset><text>Localization of Lst1p</text><annotation id="Team286_A573"><infon key="type">Gene</infon><location offset="42056" length="5"></location><text>Lst1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>42062</offset><sentence><offset>42062</offset><text>To examine the intracellular distribution of Lst1p, an epitope-tagged derivative was constructed by inserting six copies of the 10-amino acid HA near the NH2 terminus of Lst1p.</text><annotation id="Team286_A574"><infon key="type">Gene</infon><location offset="42107" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A575"><infon key="type">Gene</infon><location offset="42235" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>42242</offset><text>The HA-tagged LST1 was functional, as demonstrated by its ability to complement lst1-1 in a sectoring assay, and to restore the ability of a lst1Delta mutant to grow on acidic medium at 37 C (not shown).</text><annotation id="Team286_A576"><infon key="type">Gene</infon><location offset="42256" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A577"><infon key="type">Gene</infon><location offset="42311" length="17"></location><text>complement lst1-1</text></annotation><annotation id="Team286_A578"><infon key="type">Gene</infon><location offset="42383" length="16"></location><text>lst1Delta mutant</text></annotation></sentence><sentence><offset>42447</offset><text>In cells expressing Lst1p-HA that were fixed for immunofluorescence microscopy, staining was found primarily at the nuclear periphery (Fig. 8).</text><annotation id="Team286_A579"><infon key="type">Gene</infon><location offset="42468" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>42592</offset><text>No signal was seen in cells expressing untagged Lst1p, verifying that the origin of the staining pattern was due to Lst1p-HA.</text><annotation id="Team286_A580"><infon key="type">Gene</infon><location offset="42640" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A581"><infon key="type">Gene</infon><location offset="42709" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>42719</offset><text>Although Lst1p-HA staining largely coincided with the ER marker Kar2p, there were subtle differences in their patterns of localization: Kar2p appeared uniformly, distributed around the nuclear periphery, whereas Lst1p-HA staining had a more punctate appearance indicating that Lst1p might be concentrated in particular regions of the ER.</text><annotation id="Team286_A582"><infon key="type">Gene</infon><location offset="42728" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A583"><infon key="type">Gene</infon><location offset="42773" length="15"></location><text>ER marker Kar2p</text></annotation><annotation id="Team286_A584"><infon key="type">Gene</infon><location offset="42857" length="5"></location><text>Kar2p</text></annotation><annotation id="Team286_A585"><infon key="type">Gene</infon><location offset="42934" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A586"><infon key="type">Gene</infon><location offset="42999" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>43060</offset><text>In addition, weak punctate staining was observed throughout the cell body, some of which may correspond to ER membranes near the cell periphery.</text><annotation id="Team286_A587"><infon key="type">Gene</infon><location offset="43169" length="2"></location><text>ER</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>43209</offset><sentence><offset>43209</offset><text>The intracellular distribution of Lst1p was also examined by subcellular fractionation.</text><annotation id="Team286_A588"><infon key="type">Gene</infon><location offset="43243" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>43297</offset><text>Cells expressing Lst1p-HA were converted to spheroplasts and then gently lysed.</text><annotation id="Team286_A589"><infon key="type">Gene</infon><location offset="43314" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>43378</offset><text>This cell lysate was subjected to differential centrifugation and most of Lst1p-HA was found to pellet at either 500 g or 10,000 g (Fig. 9 A).</text><annotation id="Team286_A590"><infon key="type">Gene</infon><location offset="43453" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>43523</offset><text>All of the soluble marker protein Gdh2p (Miller and Magasanik, 1990) was found in the 150,000 g supernatant fraction, indicating complete cell lysis (data not shown).</text></sentence><sentence><offset>43692</offset><text>The association of the Lst1p protein with the sedimenting fraction was analyzed by chemical treatment of cell lysates before centrifugation at 50,000 g.</text><annotation id="Team286_A591"><infon key="type">Gene</infon><location offset="43715" length="13"></location><text>Lst1p protein</text></annotation></sentence><sentence><offset>43848</offset><text>Incubation of cell extracts in 1% Triton X-100, 2.5 M urea, 100 mM sodium carbonate, pH 11.5, or 500 mM NaCl resulted in the release of a portion of the Lst1p-HA into the soluble fraction (Fig. 9 B).</text><annotation id="Team286_A592"><infon key="type">Gene</infon><location offset="44002" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A593"><infon key="type">Gene</infon><location offset="44039" length="8"></location><text>Fig. 9 B</text></annotation></sentence><sentence><offset>44050</offset><text>The partial dissociation of Lst1p-HA from the sedimenting fraction by these agents suggested that Lst1p is a peripheral membrane protein that adheres tightly to the membrane.</text><annotation id="Team286_A594"><infon key="type">Gene</infon><location offset="44079" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A595"><infon key="type">Gene</infon><location offset="44150" length="5"></location><text>Lst1p</text></annotation></sentence></passage><passage><infon key="type">title_2</infon><offset>44229</offset><sentence><offset>44229</offset><text>Lst1p Binds Sec23p</text><annotation id="Team286_A596"><infon key="type">Gene</infon><location offset="44229" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A597"><infon key="type">Gene</infon><location offset="44241" length="6"></location><text>Sec23p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>44248</offset><sentence><offset>44248</offset><text>Sec24p was first identified as a protein that formed a 400-kD complex with Sec23p (Hicke et al., 1992).</text><annotation id="Team286_A598"><infon key="type">Gene</infon><location offset="44248" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A599"><infon key="type">Gene</infon><location offset="44324" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>44353</offset><text>Because of the similarity of Lst1p to Sec24p, we investigated whether Lst1p could also bind to Sec23p.</text><annotation id="Team286_A600"><infon key="type">Gene</infon><location offset="44383" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A601"><infon key="type">Gene</infon><location offset="44392" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A602"><infon key="type">Gene</infon><location offset="44425" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A603"><infon key="type">Gene</infon><location offset="44450" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>44458</offset><text>To assay potential interactions by the yeast two-hybrid assay, LST1 was fused to the lexA DNA-binding domain (pKR37) and SEC23 was fused to an acidic activation domain (pPE81).</text><annotation id="Team286_A604"><infon key="type">Gene</infon><location offset="44521" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A605"><infon key="type">Gene</infon><location offset="44544" length="23"></location><text>lexA DNA-binding domain</text></annotation><annotation id="Team286_A606"><infon key="type">Gene</infon><location offset="44569" length="5"></location><text>pKR37</text></annotation><annotation id="Team286_A607"><infon key="type">Gene</infon><location offset="44580" length="5"></location><text>SEC23</text></annotation></sentence><sentence><offset>44637</offset><text>Interaction between the two fusion proteins was tested by assaying for activation of a lacZ reporter gene.</text><annotation id="Team286_A608"><infon key="type">Gene</infon><location offset="44726" length="18"></location><text>lacZ reporter gene</text></annotation></sentence><sentence><offset>44746</offset><text>Induction of beta-galactosidase was observed when the LST1 and SEC23 fusions were coexpressed, but not when expressed alone (Table IV).</text><annotation id="Team286_A609"><infon key="type">Gene</infon><location offset="44759" length="18"></location><text>beta-galactosidase</text></annotation><annotation id="Team286_A610"><infon key="type">Gene</infon><location offset="44800" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A611"><infon key="type">Gene</infon><location offset="44809" length="5"></location><text>SEC23</text></annotation></sentence><sentence><offset>44884</offset><text>The level of induction caused by interaction of LST1 and SEC23 was similar to that seen for interaction of SEC24 and SEC23 (Gimeno et al., 1996).</text><annotation id="Team286_A612"><infon key="type">Gene</infon><location offset="44932" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A613"><infon key="type">Gene</infon><location offset="44942" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A614"><infon key="type">Gene</infon><location offset="44993" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A615"><infon key="type">Gene</infon><location offset="45003" length="5"></location><text>SEC23</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>45031</offset><sentence><offset>45031</offset><text>To confirm the interaction between Lst1p and Sec23p, association of these proteins was examined in yeast cell extracts.</text><annotation id="Team286_A616"><infon key="type">Gene</infon><location offset="45066" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A617"><infon key="type">Gene</infon><location offset="45076" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>45152</offset><text>The coding sequence of LST-1-HA (codons 14-927) was fused to GST and expressed in yeast from the pGAL1 promoter.</text><annotation id="Team286_A618"><infon key="type">Gene</infon><location offset="45175" length="8"></location><text>LST-1-HA</text></annotation><annotation id="Team286_A619"><infon key="type">Gene</infon><location offset="45214" length="3"></location><text>GST</text></annotation><annotation id="Team286_A620"><infon key="type">Gene</infon><location offset="45250" length="15"></location><text>pGAL1 promoter</text></annotation></sentence><sentence><offset>45267</offset><text>SEC23 was also expressed from pGAL1.</text><annotation id="Team286_A621"><infon key="type">Gene</infon><location offset="45267" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A622"><infon key="type">Gene</infon><location offset="45297" length="5"></location><text>pGAL1</text></annotation></sentence><sentence><offset>45304</offset><text>Since both proteins are largely associated with intracellular membranes (Fig. 10 B), membranes prepared from cells overexpressing both Sec23p and GST-Lst1p-HA were first extracted with 600 mM NaCl to release protein complexes from the membrane, the salt extracts were clarified by centrifugation at 90,000 g, and diluted to give a final concentration of 200 mM NaCl.</text><annotation id="Team286_A623"><infon key="type">Gene</infon><location offset="45442" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A624"><infon key="type">Gene</infon><location offset="45453" length="3"></location><text>GST</text></annotation><annotation id="Team286_A625"><infon key="type">Gene</infon><location offset="45457" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>45677</offset><text>GST-Lst1p-HA was isolated from the extracts by affinity to glutathione Sepharose beads.</text><annotation id="Team286_A626"><infon key="type">Gene</infon><location offset="45677" length="3"></location><text>GST</text></annotation><annotation id="Team286_A627"><infon key="type">Gene</infon><location offset="45681" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>45767</offset><text>Sec23p was found in association with GST-Lst1p-Ha, but not in control extracts prepared from cells expressing Sec23p and GST alone (Fig. 10 A).</text><annotation id="Team286_A628"><infon key="type">Gene</infon><location offset="45767" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A629"><infon key="type">Gene</infon><location offset="45805" length="3"></location><text>GST</text></annotation><annotation id="Team286_A630"><infon key="type">Gene</infon><location offset="45809" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A631"><infon key="type">Gene</infon><location offset="45815" length="2"></location><text>Ha</text></annotation><annotation id="Team286_A632"><infon key="type">Gene</infon><location offset="45879" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A633"><infon key="type">Gene</infon><location offset="45890" length="3"></location><text>GST</text></annotation></sentence><sentence><offset>45913</offset><text>Together, these experiments show that Lst1p, like Sec24p, can form a complex with Sec23p.</text><annotation id="Team286_A634"><infon key="type">Gene</infon><location offset="45951" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A635"><infon key="type">Gene</infon><location offset="45963" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A636"><infon key="type">Gene</infon><location offset="45996" length="6"></location><text>Sec23p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>46013</offset><sentence><offset>46013</offset><text>Sec23p and Sec24p have been shown to assemble onto the ER membrane as a complex (Matsuoka et al., 1998).</text><annotation id="Team286_A637"><infon key="type">Gene</infon><location offset="46013" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A638"><infon key="type">Gene</infon><location offset="46024" length="6"></location><text>Sec24p</text></annotation></sentence><sentence><offset>46120</offset><text>While working out conditions to optimize recovery of Sec23p bound to GST-Lst1p-HA, we discovered that assembly of an Lst1p/Sec23p complex appears to enhance the association of both proteins with the ER membrane.</text><annotation id="Team286_A639"><infon key="type">Gene</infon><location offset="46174" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A640"><infon key="type">Gene</infon><location offset="46190" length="3"></location><text>GST</text></annotation><annotation id="Team286_A641"><infon key="type">Gene</infon><location offset="46194" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A642"><infon key="type">Gene</infon><location offset="46200" length="2"></location><text>HA</text></annotation><annotation id="Team286_A643"><infon key="type">Gene</infon><location offset="46238" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A644"><infon key="type">Gene</infon><location offset="46244" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>46335</offset><text>When both GST-Lst1p-HA and Sec23p were overexpressed in the same cell, &gt;60% of the Sec23p, and 70% of the GST-Lst1p-HA were found in a fraction that pelleted at 10,000 g (Fig. 10 B).</text><annotation id="Team286_A645"><infon key="type">Gene</infon><location offset="46345" length="3"></location><text>GST</text></annotation><annotation id="Team286_A646"><infon key="type">Gene</infon><location offset="46349" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A647"><infon key="type">Gene</infon><location offset="46355" length="2"></location><text>HA</text></annotation><annotation id="Team286_A648"><infon key="type">Gene</infon><location offset="46362" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A649"><infon key="type">Gene</infon><location offset="46418" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A650"><infon key="type">Gene</infon><location offset="46442" length="3"></location><text>GST</text></annotation><annotation id="Team286_A651"><infon key="type">Gene</infon><location offset="46446" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A652"><infon key="type">Gene</infon><location offset="46452" length="2"></location><text>HA</text></annotation></sentence><sentence><offset>46520</offset><text>This pellet contains most of the ER, as marked by the ER membrane protein Sec61p (data not shown).</text><annotation id="Team286_A653"><infon key="type">Gene</infon><location offset="46575" length="27"></location><text>ER membrane protein Sec61p</text></annotation></sentence><sentence><offset>46621</offset><text>When material that pelleted at 10,000 g was suspended in 60% sucrose and applied to the bottom of a sucrose density gradient, &gt;90% of the GST- Lst1p-HA and Sec23p cofractionated with the ER resident membrane protein, Sec61p, at a density corresponding to 45% sucrose, showing that GST-Lst1p-HA and Sec23p were associated with membranes (data not shown).</text><annotation id="Team286_A654"><infon key="type">Gene</infon><location offset="46761" length="3"></location><text>GST</text></annotation><annotation id="Team286_A655"><infon key="type">Gene</infon><location offset="46766" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A656"><infon key="type">Gene</infon><location offset="46772" length="2"></location><text>HA</text></annotation><annotation id="Team286_A657"><infon key="type">Gene</infon><location offset="46779" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A658"><infon key="type">Gene</infon><location offset="46810" length="29"></location><text>ER resident membrane protein</text></annotation><annotation id="Team286_A659"><infon key="type">Gene</infon><location offset="46841" length="6"></location><text>Sec61p</text></annotation><annotation id="Team286_A660"><infon key="type">Gene</infon><location offset="46906" length="3"></location><text>GST</text></annotation><annotation id="Team286_A661"><infon key="type">Gene</infon><location offset="46910" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A662"><infon key="type">Gene</infon><location offset="46916" length="2"></location><text>HA</text></annotation><annotation id="Team286_A663"><infon key="type">Gene</infon><location offset="46923" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>46980</offset><text>In contrast to the case when Sec23p and GST-Lst1p-HA were expressed together, &lt;10% of the Sec23p pelleted at 10,000 g in lysates from a strain overexpressing Sec23p alone.</text><annotation id="Team286_A664"><infon key="type">Gene</infon><location offset="47009" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A665"><infon key="type">Gene</infon><location offset="47020" length="3"></location><text>GST</text></annotation><annotation id="Team286_A666"><infon key="type">Gene</infon><location offset="47024" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A667"><infon key="type">Gene</infon><location offset="47030" length="2"></location><text>HA</text></annotation><annotation id="Team286_A668"><infon key="type">Gene</infon><location offset="47071" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A669"><infon key="type">Gene</infon><location offset="47140" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>47154</offset><text>Similarly, &lt;20% of the GST-Lst1p-HA pelleted at 10,000 g in lysates from a strain expressing GST-Lst1p-HA alone (Fig. 10 B).</text><annotation id="Team286_A670"><infon key="type">Gene</infon><location offset="47177" length="3"></location><text>GST</text></annotation><annotation id="Team286_A671"><infon key="type">Gene</infon><location offset="47181" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A672"><infon key="type">Gene</infon><location offset="47187" length="2"></location><text>HA</text></annotation><annotation id="Team286_A673"><infon key="type">Gene</infon><location offset="47248" length="3"></location><text>GST</text></annotation><annotation id="Team286_A674"><infon key="type">Gene</infon><location offset="47252" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>47281</offset><text>Thus, when either Sec23p or GST-Lst1p-HA was overexpressed alone, most of the overexpressed protein was soluble, but when the proteins were expressed together, most of the proteins were associated with the ER membranes.</text><annotation id="Team286_A675"><infon key="type">Gene</infon><location offset="47299" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A676"><infon key="type">Gene</infon><location offset="47309" length="3"></location><text>GST</text></annotation><annotation id="Team286_A677"><infon key="type">Gene</infon><location offset="47313" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A678"><infon key="type">Gene</infon><location offset="47319" length="2"></location><text>HA</text></annotation></sentence><sentence><offset>47503</offset><text>These data support the observation that Lst1p can form a complex with Sec23p, and that the Lst1p/ Sec23p complex has affinity for ER membranes.</text><annotation id="Team286_A679"><infon key="type">Gene</infon><location offset="47543" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A680"><infon key="type">Gene</infon><location offset="47574" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A681"><infon key="type">Gene</infon><location offset="47595" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A682"><infon key="type">Gene</infon><location offset="47602" length="6"></location><text>Sec23p</text></annotation></sentence></passage><passage><infon key="type">title_1</infon><offset>47666</offset><sentence><offset>47666</offset><text>Discussion</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>47677</offset><sentence><offset>47677</offset><text>By screening for mutants that exhibited synthetic-lethal genetic interactions with the COPII mutation sec13-1, we identified the LST1 gene.</text><annotation id="Team286_A683"><infon key="type">Gene</infon><location offset="47765" length="22"></location><text>COPII mutation sec13-1</text></annotation><annotation id="Team286_A684"><infon key="type">Gene</infon><location offset="47808" length="9"></location><text>LST1 gene</text></annotation></sentence><sentence><offset>47819</offset><text>Subsequent genetic tests showed that lst1Delta is lethal when combined with mutations in genes required for COPII vesicle budding from the ER (SEC12, SEC13, SEC16, SEC23, SEC24, and SEC31), but lst1Delta is not lethal when combined with mutations in genes that are required for vesicle fusion with the Golgi compartment (SEC17 and SEC18).</text><annotation id="Team286_A685"><infon key="type">Gene</infon><location offset="47857" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A686"><infon key="type">Gene</infon><location offset="47960" length="2"></location><text>ER</text></annotation><annotation id="Team286_A687"><infon key="type">Gene</infon><location offset="47964" length="5"></location><text>SEC12</text></annotation><annotation id="Team286_A688"><infon key="type">Gene</infon><location offset="47972" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A689"><infon key="type">Gene</infon><location offset="47979" length="5"></location><text>SEC16</text></annotation><annotation id="Team286_A690"><infon key="type">Gene</infon><location offset="47986" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A691"><infon key="type">Gene</infon><location offset="47993" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A692"><infon key="type">Gene</infon><location offset="48004" length="5"></location><text>SEC31</text></annotation><annotation id="Team286_A693"><infon key="type">Gene</infon><location offset="48146" length="5"></location><text>SEC17</text></annotation><annotation id="Team286_A694"><infon key="type">Gene</infon><location offset="48156" length="5"></location><text>SEC18</text></annotation></sentence><sentence><offset>48164</offset><text>This pattern of genetic interactions indicated that LST1 participates in the process of vesicle budding from the ER, an expectation that was born out by the examination of the LST1 gene and its product.</text><annotation id="Team286_A695"><infon key="type">Gene</infon><location offset="48217" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A696"><infon key="type">Gene</infon><location offset="48343" length="9"></location><text>LST1 gene</text></annotation></sentence><sentence><offset>48370</offset><text>The following observations indicate a role for Lst1p as part of a COPII-like vesicle coat: (I) LST1 encodes a 90-kD protein that is homologous to the COPII-coat subunit Sec24p.</text><annotation id="Team286_A697"><infon key="type">Gene</infon><location offset="48417" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A698"><infon key="type">Gene</infon><location offset="48466" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A699"><infon key="type">Gene</infon><location offset="48522" length="25"></location><text>COPII-coat subunit Sec24p</text></annotation></sentence><sentence><offset>48549</offset><text>The two proteins share 23% amino acid identity over their entire lengths.</text></sentence><sentence><offset>48624</offset><text>(II) Lst1p is a peripheral ER membrane protein as shown by immunofluorescence microscopy and cell fractionation.</text><annotation id="Team286_A700"><infon key="type">Gene</infon><location offset="48629" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A701"><infon key="type">Gene</infon><location offset="48651" length="19"></location><text>ER membrane protein</text></annotation></sentence><sentence><offset>48738</offset><text>(III) Lst1p, like Sec24p, can bind to Sec23p as shown by tests for two-hybrid interaction and affinity purification of a complex of GST-Lst1p and Sec23p.</text><annotation id="Team286_A702"><infon key="type">Gene</infon><location offset="48744" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A703"><infon key="type">Gene</infon><location offset="48756" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A704"><infon key="type">Gene</infon><location offset="48776" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A705"><infon key="type">Gene</infon><location offset="48871" length="3"></location><text>GST</text></annotation><annotation id="Team286_A706"><infon key="type">Gene</infon><location offset="48875" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A707"><infon key="type">Gene</infon><location offset="48885" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>48894</offset><text>(IV) Assembly of the Sec23p-Lst1p complex appears to enhance the membrane association of both Lst1p and Sec23p: when both proteins are overexpressed together, most associate with membranes, whereas either protein overexpressed alone is mostly cytosolic.</text><annotation id="Team286_A708"><infon key="type">Gene</infon><location offset="48915" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A709"><infon key="type">Gene</infon><location offset="48922" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A710"><infon key="type">Gene</infon><location offset="48989" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A711"><infon key="type">Gene</infon><location offset="49000" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>49152</offset><text>(V) Although strains with chromosomal deletion of LST1 are viable and appear normal for secretion of marker proteins, these mutants show a pronounced accumulation of Pma1p in the ER, indicating a selective defect in ER to Golgi traffic.</text><annotation id="Team286_A712"><infon key="type">Gene</infon><location offset="49203" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A713"><infon key="type">Gene</infon><location offset="49320" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A714"><infon key="type">Gene</infon><location offset="49334" length="2"></location><text>ER</text></annotation></sentence><sentence><offset>49393</offset><text>Based on these findings, we propose that Lst1p takes the place of Sec24p in a specialized COPII coat complex that is used for the recruitment of Pma1p into vesicles.</text><annotation id="Team286_A715"><infon key="type">Gene</infon><location offset="49434" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A716"><infon key="type">Gene</infon><location offset="49460" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A717"><infon key="type">Gene</infon><location offset="49484" length="18"></location><text>COPII coat complex</text></annotation><annotation id="Team286_A718"><infon key="type">Gene</infon><location offset="49540" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>49559</offset><sentence><offset>49559</offset><text>Strains carrying lst1Delta have the phenotypic hallmarks of a deficiency in Pma1p activity, including sensitivity to growth in an acidic environment, the formation of multibudded cells, and a decreased rate of proton efflux from intact cells.</text><annotation id="Team286_A719"><infon key="type">Gene</infon><location offset="49576" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A720"><infon key="type">Gene</infon><location offset="49636" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>49805</offset><text>All three traits are expressed only at temperatures of 30 C and above, indicating that LST1 is only required for Pma1p activity at high temperature.</text><annotation id="Team286_A721"><infon key="type">Gene</infon><location offset="49892" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A722"><infon key="type">Gene</infon><location offset="49918" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>49954</offset><text>Localization of Pma1p in lst1Delta cells by immunofluorescence and sucrose density cell fractionation demonstrate that the transport of Pma1p from the ER is compromised in lst1Delta at 37 C.</text><annotation id="Team286_A723"><infon key="type">Gene</infon><location offset="49970" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A724"><infon key="type">Gene</infon><location offset="50092" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A725"><infon key="type">Gene</infon><location offset="50107" length="2"></location><text>ER</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>50141</offset><sentence><offset>50141</offset><text>Export of Pma1p from the ER cannot be completely dependent on Lst1p, since Pma1p transport appears normal in lst1Delta mutants at 24 C.</text><annotation id="Team286_A726"><infon key="type">Gene</infon><location offset="50151" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A727"><infon key="type">Gene</infon><location offset="50203" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A728"><infon key="type">Gene</infon><location offset="50216" length="15"></location><text>Pma1p transport</text></annotation></sentence><sentence><offset>50278</offset><text>Even at 37 C, the block in Pma1p transport may not be complete since ~35% of the total Pma1p fractionates with the plasma membrane, although some of the Pma1p detected in the plasma membrane in this experiment was probably synthesized before the shift to restrictive temperature.</text><annotation id="Team286_A729"><infon key="type">Gene</infon><location offset="50305" length="16"></location><text>Pma1p transport</text></annotation><annotation id="Team286_A730"><infon key="type">Gene</infon><location offset="50367" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A731"><infon key="type">Gene</infon><location offset="50434" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>50563</offset><text>Therefore, it seems likely that Lst1p and Sec24p share the burden of transporting Pma1p from the ER.</text><annotation id="Team286_A732"><infon key="type">Gene</infon><location offset="50596" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A733"><infon key="type">Gene</infon><location offset="50606" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A734"><infon key="type">Gene</infon><location offset="50646" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>50666</offset><text>At 24 C, it appears that Sec24p (or some other protein) can compensate for the absence of Lst1p, but at temperatures of 30 C or higher, compensation is no longer possible unless extra copies of Sec24p are provided by expression from a multicopy plasmid.</text><annotation id="Team286_A735"><infon key="type">Gene</infon><location offset="50691" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A736"><infon key="type">Gene</infon><location offset="50757" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A737"><infon key="type">Gene</infon><location offset="50863" length="6"></location><text>Sec24p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>50927</offset><sentence><offset>50927</offset><text>The transport defect caused by deletion of LST1 appears to be specific for Pma1p.</text><annotation id="Team286_A738"><infon key="type">Gene</infon><location offset="50970" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A739"><infon key="type">Gene</infon><location offset="51002" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>51009</offset><text>Under conditions where a defect in Pma1p transport was observed in lst1Delta mutants, transport of Gas1p, carboxypeptidase Y, and invertase was unaffected.</text><annotation id="Team286_A740"><infon key="type">Gene</infon><location offset="51045" length="15"></location><text>Pma1p transport</text></annotation><annotation id="Team286_A741"><infon key="type">Gene</infon><location offset="51077" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A742"><infon key="type">Gene</infon><location offset="51110" length="5"></location><text>Gas1p</text></annotation><annotation id="Team286_A743"><infon key="type">Gene</infon><location offset="51117" length="18"></location><text>carboxypeptidase Y</text></annotation><annotation id="Team286_A744"><infon key="type">Gene</infon><location offset="51141" length="9"></location><text>invertase</text></annotation></sentence><sentence><offset>51168</offset><text>Using growth as a more general assay for trafficking defects, we found that lst1Delta mutants grew at an identical rate to wild-type at 37 C when we compensated for the defect in Pma1p transport by using media at pH 6.5.</text><annotation id="Team286_A745"><infon key="type">Gene</infon><location offset="51245" length="17"></location><text>lst1Delta mutants</text></annotation><annotation id="Team286_A746"><infon key="type">Gene</infon><location offset="51350" length="15"></location><text>Pma1p transport</text></annotation></sentence><sentence><offset>51393</offset><text>This indicates that rate of expansion of the plasma membrane, including the transport of all essential plasma membrane proteins, is not significantly affected by the absence of LST1.</text><annotation id="Team286_A747"><infon key="type">Gene</infon><location offset="51572" length="4"></location><text>LST1</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>51573</offset><sentence><offset>51573</offset><text>We also considered the possibility that there may be differences among cargo molecules in their response to general defects in the protein transport machinery.</text></sentence><sentence><offset>51733</offset><text>Of particular concern was the possibility that Pma1p transport might be particularly sensitive to slowed ER to Golgi transport, such that a defect in transport too subtle to have an effect on our standard marker proteins might have a significant effect on the rate of transport of Pma1p.</text><annotation id="Team286_A748"><infon key="type">Gene</infon><location offset="51780" length="15"></location><text>Pma1p transport</text></annotation><annotation id="Team286_A749"><infon key="type">Gene</infon><location offset="52018" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>52025</offset><text>If this were the case, partial defects in other COPII components should also interfere with Pma1p transport.</text><annotation id="Team286_A750"><infon key="type">Gene</infon><location offset="52119" length="15"></location><text>Pma1p transport</text></annotation></sentence><sentence><offset>52136</offset><text>Therefore, we examined sec24 and sec31 mutants, but could find no evidence for a defect in Pma1p transport, even at semipermissive temperatures where the rate of growth was inhibited.</text><annotation id="Team286_A751"><infon key="type">Gene</infon><location offset="52159" length="5"></location><text>sec24</text></annotation><annotation id="Team286_A752"><infon key="type">Gene</infon><location offset="52169" length="13"></location><text>sec31 mutants</text></annotation><annotation id="Team286_A753"><infon key="type">Gene</infon><location offset="52228" length="15"></location><text>Pma1p transport</text></annotation></sentence><sentence><offset>52322</offset><text>Although Pma1p was the only essential protein for which we could detect a transport defect in lst1 mutants, a defect in the transport of any nonessential protein could have been overlooked by our analysis.</text><annotation id="Team286_A754"><infon key="type">Gene</infon><location offset="52331" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A755"><infon key="type">Gene</infon><location offset="52417" length="12"></location><text>lst1 mutants</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>52531</offset><sentence><offset>52531</offset><text>Factors required for the transport of specific membrane proteins have been documented in a number of other cases.</text></sentence><sentence><offset>52647</offset><text>The SHR3 gene encodes an ER resident protein that is required for the transport of amino acid permeases out of the ER, but is not required for the transport of a variety of other proteins (Ljungdahl et al., 1992; Kuehn et al., 1996).</text><annotation id="Team286_A756"><infon key="type">Gene</infon><location offset="52651" length="9"></location><text>SHR3 gene</text></annotation><annotation id="Team286_A757"><infon key="type">Gene</infon><location offset="52672" length="2"></location><text>ER</text></annotation><annotation id="Team286_A758"><infon key="type">Gene</infon><location offset="52764" length="2"></location><text>ER</text></annotation></sentence><sentence><offset>52884</offset><text>A set of ER proteins, Vma12p, Vma21p, and Vma22p, are required for transport from the ER of the integral membrane subunit of the vacuolar ATPase (Hill and Stevens, 1994, 1995; Jackson and Stevens, 1997).</text><annotation id="Team286_A759"><infon key="type">Gene</infon><location offset="52893" length="11"></location><text>ER proteins</text></annotation><annotation id="Team286_A760"><infon key="type">Gene</infon><location offset="52906" length="6"></location><text>Vma12p</text></annotation><annotation id="Team286_A761"><infon key="type">Gene</infon><location offset="52914" length="6"></location><text>Vma21p</text></annotation><annotation id="Team286_A762"><infon key="type">Gene</infon><location offset="52927" length="6"></location><text>Vma22p</text></annotation><annotation id="Team286_A763"><infon key="type">Gene</infon><location offset="53014" length="15"></location><text>vacuolar ATPase</text></annotation></sentence><sentence><offset>53090</offset><text>Similarly, mutational studies have shown that the small ER membrane protein Erv14p is specifically required for transport of the plasma membrane protein Axl2p out of the ER (Powers and Barlowe, 1998).</text><annotation id="Team286_A764"><infon key="type">Gene</infon><location offset="53140" length="33"></location><text>small ER membrane protein Erv14p</text></annotation><annotation id="Team286_A765"><infon key="type">Gene</infon><location offset="53221" length="29"></location><text>plasma membrane protein Axl2p</text></annotation></sentence><sentence><offset>53294</offset><text>Finally, Ast1p has been suggested to be a factor specifically needed for the transport of Pma1p from the Golgi compartment to the plasma membrane (Chang and Fink, 1995).</text><annotation id="Team286_A766"><infon key="type">Gene</infon><location offset="53303" length="5"></location><text>Ast1p</text></annotation><annotation id="Team286_A767"><infon key="type">Gene</infon><location offset="53386" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>53467</offset><text>In all of these cases, the question remains whether Shr3p, the Vma proteins, Erv14p, or Ast1p act directly in vesicular transport of their respective cargo molecules, or whether they are primarily involved in protein folding and influence protein sorting indirectly through quality control mechanisms.</text><annotation id="Team286_A768"><infon key="type">Gene</infon><location offset="53520" length="5"></location><text>Shr3p</text></annotation><annotation id="Team286_A769"><infon key="type">Gene</infon><location offset="53531" length="12"></location><text>Vma proteins</text></annotation><annotation id="Team286_A770"><infon key="type">Gene</infon><location offset="53545" length="6"></location><text>Erv14p</text></annotation><annotation id="Team286_A771"><infon key="type">Gene</infon><location offset="53556" length="5"></location><text>Ast1p</text></annotation></sentence><sentence><offset>53772</offset><text>Because Lst1p appears to be a component of a vesicle coat, Lst1p seems more likely to have a direct role in the sorting of Pma1p rather than in its folding.</text><annotation id="Team286_A772"><infon key="type">Gene</infon><location offset="53780" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A773"><infon key="type">Gene</infon><location offset="53832" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A774"><infon key="type">Gene</infon><location offset="53897" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>53931</offset><sentence><offset>53931</offset><text>Expression of a variety of dominant PMA1 mutations can cause accumulation of both mutant and wild-type Pma1p in proliferated ER (Harris et al., 1994; Portillo, 1997).</text><annotation id="Team286_A775"><infon key="type">Gene</infon><location offset="53958" length="23"></location><text>dominant PMA1 mutations</text></annotation><annotation id="Team286_A776"><infon key="type">Gene</infon><location offset="54036" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>54101</offset><text>Similarly, the transport of wild-type Pma1p from the ER is blocked when PMA2 (an isoform of PMA1) or plant plasma membrane proton-ATPases are overexpressed in yeast (Villalba et al., 1992; Supply et al., 1994; de Kerchove d'Exaerde et al., 1995).</text><annotation id="Team286_A777"><infon key="type">Gene</infon><location offset="54139" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A778"><infon key="type">Gene</infon><location offset="54194" length="4"></location><text>PMA1</text></annotation><annotation id="Team286_A779"><infon key="type">Gene</infon><location offset="54233" length="7"></location><text>ATPases</text></annotation></sentence><sentence><offset>54351</offset><text>One proposal was that a special factor may be required for the transport of Pma1p from the ER in a manner analogous to the requirement for Shr3p in the transport of amino acid permeases (Supply et al., 1994).</text><annotation id="Team286_A780"><infon key="type">Gene</infon><location offset="54429" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A781"><infon key="type">Gene</infon><location offset="54492" length="5"></location><text>Shr3p</text></annotation></sentence><sentence><offset>54563</offset><text>The specific role of Lst1p in the transport of Pma1p suggests that it may be the factor depleted by the expression of dominant forms of Pma1p.</text><annotation id="Team286_A782"><infon key="type">Gene</infon><location offset="54584" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A783"><infon key="type">Gene</infon><location offset="54611" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A784"><infon key="type">Gene</infon><location offset="54700" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>54707</offset><text>In the future, it may be possible to test this idea by evaluating the ability of Lst1p overexpression to reverse the effects of dominant PMA1 mutations.</text><annotation id="Team286_A785"><infon key="type">Gene</infon><location offset="54790" length="5"></location><text>Lst1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>54863</offset><sentence><offset>54863</offset><text>The mechanism by which Lst1p acts in the transport of Pma1p may be inferred from recent studies examining the recruitment of cargo molecules into COPII vesicles.</text><annotation id="Team286_A786"><infon key="type">Gene</infon><location offset="54886" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A787"><infon key="type">Gene</infon><location offset="54918" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>55027</offset><text>Using ER-derived microsomes and purified COPII components, Kuehn et al. (1998) have shown that the Sec23p/Sec24p complex, along with Sar1p, associate with amino acid permeases and other integral membrane protein that are destined for the plasma membrane.</text><annotation id="Team286_A788"><infon key="type">Gene</infon><location offset="55128" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A789"><infon key="type">Gene</infon><location offset="55135" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A790"><infon key="type">Gene</infon><location offset="55163" length="5"></location><text>Sar1p</text></annotation></sentence><sentence><offset>55285</offset><text>In parallel experiments using mammalian microsomes, mammalian Sec23p/Sec24p and Sar1p were found to bind to microsomal membranes and form a complex that contains the cargo protein VSV-G (Aridor et al., 1998).</text><annotation id="Team286_A791"><infon key="type">Gene</infon><location offset="55348" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A792"><infon key="type">Gene</infon><location offset="55355" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A793"><infon key="type">Gene</infon><location offset="55367" length="5"></location><text>Sar1p</text></annotation></sentence><sentence><offset>55498</offset><text>The conclusion from both experimental systems is that the Sec23p/Sec24p complex contains specific binding sites for the capture of membrane cargo proteins within the plane of the ER membrane.</text><annotation id="Team286_A794"><infon key="type">Gene</infon><location offset="55556" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A795"><infon key="type">Gene</infon><location offset="55563" length="6"></location><text>Sec24p</text></annotation></sentence><sentence><offset>55692</offset><text>Based on the data presented here, Lst1p appears to be an isoform of Sec24p that is adapted for selection of Pma1p.</text><annotation id="Team286_A796"><infon key="type">Gene</infon><location offset="55726" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A797"><infon key="type">Gene</infon><location offset="55761" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A798"><infon key="type">Gene</infon><location offset="55801" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>55809</offset><text>This provides the first evidence that Sec24 family members carry information specifying the type of cargo molecules that are accepted by ER-derived vesicles.</text><annotation id="Team286_A799"><infon key="type">Gene</infon><location offset="55847" length="12"></location><text>Sec24 family</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>55967</offset><sentence><offset>55967</offset><text>We have looked for association of Lst1p with ER-derived vesicles, but under the conditions of an in vitro budding reaction, a large quantity of Lst1p-HA is released from the membrane in soluble form.</text><annotation id="Team286_A800"><infon key="type">Gene</infon><location offset="56001" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A801"><infon key="type">Gene</infon><location offset="56112" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>56169</offset><text>Soluble Lst1p-HA gives a high background in vesicle fractions preventing us from reliably determining whether there is a specific association of Lst1p with vesicles.</text><annotation id="Team286_A802"><infon key="type">Gene</infon><location offset="56177" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A803"><infon key="type">Gene</infon><location offset="56316" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>56337</offset><text>In future experiments, it may be possible to isolate vesicles coated with Lst1p by performing an in vitro budding reaction using purified cytosolic components, including a purified complex of Lst1p and Sec23p.</text><annotation id="Team286_A804"><infon key="type">Gene</infon><location offset="56412" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A805"><infon key="type">Gene</infon><location offset="56531" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A806"><infon key="type">Gene</infon><location offset="56542" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>56550</offset><text>It may also be possible to determine whether vesicles that are formed using a Sec23p/Lst1p complex more efficiently incorporate Pma1p than vesicles formed using the Sec23p/Sec24p complex.</text><annotation id="Team286_A807"><infon key="type">Gene</infon><location offset="56629" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A808"><infon key="type">Gene</infon><location offset="56636" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A809"><infon key="type">Gene</infon><location offset="56680" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A810"><infon key="type">Gene</infon><location offset="56718" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A811"><infon key="type">Gene</infon><location offset="56725" length="6"></location><text>Sec24p</text></annotation></sentence><sentence><offset>56741</offset><text>Finally, it will be of interest to determine if there is direct binding of Lst1p to Pma1p.</text><annotation id="Team286_A812"><infon key="type">Gene</infon><location offset="56816" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A813"><infon key="type">Gene</infon><location offset="56826" length="5"></location><text>Pma1p</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>56833</offset><sentence><offset>56833</offset><text>The identification of a Sec24p homologue that also acts in transport from the ER raises the possibility that the coats of ER-derived vesicles may be heterogeneous.</text><annotation id="Team286_A814"><infon key="type">Gene</infon><location offset="56857" length="6"></location><text>Sec24p</text></annotation></sentence><sentence><offset>56999</offset><text>It is possible that Sec23p/Lst1p complexes act to form a class of vesicle that is distinct from those formed by Sec23p/ Sec24p complexes.</text><annotation id="Team286_A815"><infon key="type">Gene</infon><location offset="57020" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A816"><infon key="type">Gene</infon><location offset="57027" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A817"><infon key="type">Gene</infon><location offset="57113" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A818"><infon key="type">Gene</infon><location offset="57121" length="6"></location><text>Sec24p</text></annotation></sentence><sentence><offset>57139</offset><text>Alternatively, it is possible that the two complexes assemble together forming vesicles with coats of mixed composition.</text></sentence><sentence><offset>57262</offset><text>The identification of additional homologues of Sec23p and Sec24p suggest the existence of coats with even greater combinatorial complexity.</text><annotation id="Team286_A819"><infon key="type">Gene</infon><location offset="57309" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A820"><infon key="type">Gene</infon><location offset="57320" length="6"></location><text>Sec24p</text></annotation></sentence><sentence><offset>57403</offset><text>We have identified a third Sec24p family member, which we call Iss1p, as a protein that binds to Sec16p.</text><annotation id="Team286_A821"><infon key="type">Gene</infon><location offset="57431" length="20"></location><text>Sec24p family member</text></annotation><annotation id="Team286_A822"><infon key="type">Gene</infon><location offset="57468" length="5"></location><text>Iss1p</text></annotation><annotation id="Team286_A823"><infon key="type">Gene</infon><location offset="57502" length="6"></location><text>Sec16p</text></annotation></sentence><sentence><offset>57510</offset><text>Iss1p (YNL049c) also binds Sec23p and appears to be associated with the ER membrane (Gimeno, 1996).</text><annotation id="Team286_A824"><infon key="type">Gene</infon><location offset="57510" length="5"></location><text>Iss1p</text></annotation><annotation id="Team286_A825"><infon key="type">Gene</infon><location offset="57518" length="7"></location><text>YNL049c</text></annotation><annotation id="Team286_A826"><infon key="type">Gene</infon><location offset="57538" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>57612</offset><text>In addition, the Saccharomyces genome contains an uncharacterized open reading frame (YHR035w) that is 21% identical to Sec23p (Saccharomyces Genome Database, Cherry et al., 1997).</text></sentence><sentence><offset>57796</offset><text>If each of the Sec23p and Sec24p homologues carry different determinants for cargo selection, and if mixed coats can form, the possible combinations of Sec23p and Sec24p homologues should allow the formation of a wide variety of COPII-like vesicles with different capacities to carry different cargo molecules.</text><annotation id="Team286_A827"><infon key="type">Gene</infon><location offset="57812" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A828"><infon key="type">Gene</infon><location offset="57823" length="6"></location><text>Sec24p</text></annotation><annotation id="Team286_A829"><infon key="type">Gene</infon><location offset="57951" length="6"></location><text>Sec23p</text></annotation><annotation id="Team286_A830"><infon key="type">Gene</infon><location offset="57962" length="6"></location><text>Sec24p</text></annotation></sentence></passage><passage><infon key="type">title_1</infon><offset>58111</offset><sentence><offset>58111</offset><text>Figures and Tables</text></sentence></passage><passage><infon key="type">table_caption</infon><offset>58130</offset><sentence><offset>58130</offset><text>S. cerevisiae Strains</text></sentence></passage><passage><infon key="type">table</infon><offset>58153</offset><sentence><offset>58156</offset><text>Strain Genotype Source or reference CUY563 MATaade2-101 ade3-24 leu2-3,112 ura3-52 T.</text><annotation id="Team286_A831"><infon key="type">Gene</infon><location offset="58196" length="35"></location><text>CUY563 MATaade2-101 ade3-24 leu2-3</text></annotation></sentence><sentence><offset>58248</offset><text>Huffaker (Cornell University) CUY564 MATalpha ade2-101 ade3-2 leu2-3,112 ura3-52 T.</text><annotation id="Team286_A832"><infon key="type">Gene</infon><location offset="58287" length="31"></location><text>MATalpha ade2-101 ade3-2 leu2-3</text></annotation></sentence><sentence><offset>58335</offset><text>Huffaker (Cornell University) EGY40 MATalpha ura3-52 leu2 his3 trp1 Golemis and Brent, 1992 CKY45 MATalpha sec13-1 his4-619 ura3-52 Kaiser Lab Collection CKY50 MATalpha sec16-2 his4-619 ura3-52 Kaiser Lab Collection CKY54 MATalpha sec17-1 his4-619 ura3-52 Kaiser Lab Collection CKY58 MATalpha sec18-1 his4-619 ura3-52 Kaiser Lab Collection CKY78 MATalpha sec23-1 his4-619 ura3-52 Kaiser Lab Collection CKY348 MATa/alpha leu2-3/leu2-3 ura3-52/ura3-52 Kaiser Lab Collection CKY423 MATalpha sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY424 MATasec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY426 MATalst1-1sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY435 MATalst1-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY436 MATalst2-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY437 MATalst3-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY438 MATalst4-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY439 MATalst5-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY440 MATalst6-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY441 MATalst7-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY442 MATalst8-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY443 MATa prototroph Kaiser Lab Collection CKY473 MATaleu2-3,112 ura3-52 Gal+ Kaiser Lab Collection CKY534 MATalpha lst1Delta::LEU2 leu2-3,112 ura3-52 [pKR17HA] CKY535 MATalst1Delta::LEU2 leu2-3,112 ura3-52 [pKR17HA] CKY536 MATalst1Delta::LEU2 ura3-52 leu2-3,112 CKY540 MATaleu2-3,112 ura3-52 [pNV31] CKY541 MATasec12-4 ura3-52 [pNV31] CKY542 MATalst1Delta::LEU2 leu2-3,112 ura3-52 [pNV31] CKY552 MATalpha lst1Delta::LEU2 leu2-3,112 ura3-52</text><annotation id="Team286_A833"><infon key="type">Gene</infon><location offset="58373" length="175"></location><text>MATalpha ura3-52 leu2 his3 trp1 Golemis and Brent, 1992 CKY45 MATalpha sec13-1 his4-619 ura3-52 Kaiser Lab Collection CKY50 MATalpha sec16-2 his4-619 ura3-52 Kaiser Lab</text></annotation><annotation id="Team286_A834"><infon key="type">Gene</infon><location offset="58561" length="52"></location><text>CKY54 MATalpha sec17-1 his4-619 ura3-52 Kaiser Lab</text></annotation><annotation id="Team286_A835"><infon key="type">Gene</infon><location offset="58626" length="117"></location><text>CKY58 MATalpha sec18-1 his4-619 ura3-52 Kaiser Lab Collection CKY78 MATalpha sec23-1 his4-619 ura3-52 Kaiser Lab</text></annotation><annotation id="Team286_A836"><infon key="type">Gene</infon><location offset="58756" length="25"></location><text>CKY348 MATa/alpha leu2-3</text></annotation><annotation id="Team286_A837"><infon key="type">Gene</infon><location offset="58782" length="14"></location><text>leu2-3 ura3-52</text></annotation><annotation id="Team286_A838"><infon key="type">Gene</infon><location offset="58797" length="158"></location><text>ura3-52 Kaiser Lab Collection CKY423 MATalpha sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY424 MATasec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52</text></annotation><annotation id="Team286_A839"><infon key="type">Gene</infon><location offset="58957" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A840"><infon key="type">Gene</infon><location offset="58966" length="61"></location><text>CKY426 MATalst1-1sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52</text></annotation><annotation id="Team286_A841"><infon key="type">Gene</infon><location offset="59029" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A842"><infon key="type">Gene</infon><location offset="59038" length="26"></location><text>CKY435 MATalst1-1 sec13-1</text></annotation><annotation id="Team286_A843"><infon key="type">Gene</infon><location offset="59067" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A844"><infon key="type">Gene</infon><location offset="59074" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A845"><infon key="type">Gene</infon><location offset="59080" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A846"><infon key="type">Gene</infon><location offset="59104" length="3"></location><text>112</text></annotation><annotation id="Team286_A847"><infon key="type">Gene</infon><location offset="59108" length="37"></location><text>ura3-52 CKY436 MATalst2-1 sec13-1</text></annotation><annotation id="Team286_A848"><infon key="type">Gene</infon><location offset="59148" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A849"><infon key="type">Gene</infon><location offset="59155" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A850"><infon key="type">Gene</infon><location offset="59161" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A851"><infon key="type">Gene</infon><location offset="59185" length="3"></location><text>112</text></annotation><annotation id="Team286_A852"><infon key="type">Gene</infon><location offset="59189" length="37"></location><text>ura3-52 CKY437 MATalst3-1 sec13-1</text></annotation><annotation id="Team286_A853"><infon key="type">Gene</infon><location offset="59229" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A854"><infon key="type">Gene</infon><location offset="59236" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A855"><infon key="type">Gene</infon><location offset="59242" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A856"><infon key="type">Gene</infon><location offset="59266" length="3"></location><text>112</text></annotation><annotation id="Team286_A857"><infon key="type">Gene</infon><location offset="59270" length="37"></location><text>ura3-52 CKY438 MATalst4-1 sec13-1</text></annotation><annotation id="Team286_A858"><infon key="type">Gene</infon><location offset="59310" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A859"><infon key="type">Gene</infon><location offset="59317" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A860"><infon key="type">Gene</infon><location offset="59323" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A861"><infon key="type">Gene</infon><location offset="59347" length="3"></location><text>112</text></annotation><annotation id="Team286_A862"><infon key="type">Gene</infon><location offset="59351" length="37"></location><text>ura3-52 CKY439 MATalst5-1 sec13-1</text></annotation><annotation id="Team286_A863"><infon key="type">Gene</infon><location offset="59391" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A864"><infon key="type">Gene</infon><location offset="59398" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A865"><infon key="type">Gene</infon><location offset="59404" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A866"><infon key="type">Gene</infon><location offset="59428" length="3"></location><text>112</text></annotation><annotation id="Team286_A867"><infon key="type">Gene</infon><location offset="59432" length="37"></location><text>ura3-52 CKY440 MATalst6-1 sec13-1</text></annotation><annotation id="Team286_A868"><infon key="type">Gene</infon><location offset="59472" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A869"><infon key="type">Gene</infon><location offset="59479" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A870"><infon key="type">Gene</infon><location offset="59485" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A871"><infon key="type">Gene</infon><location offset="59509" length="3"></location><text>112</text></annotation><annotation id="Team286_A872"><infon key="type">Gene</infon><location offset="59513" length="37"></location><text>ura3-52 CKY441 MATalst7-1 sec13-1</text></annotation><annotation id="Team286_A873"><infon key="type">Gene</infon><location offset="59553" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A874"><infon key="type">Gene</infon><location offset="59560" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A875"><infon key="type">Gene</infon><location offset="59566" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A876"><infon key="type">Gene</infon><location offset="59590" length="3"></location><text>112</text></annotation><annotation id="Team286_A877"><infon key="type">Gene</infon><location offset="59594" length="37"></location><text>ura3-52 CKY442 MATalst8-1 sec13-1</text></annotation><annotation id="Team286_A878"><infon key="type">Gene</infon><location offset="59634" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A879"><infon key="type">Gene</infon><location offset="59641" length="4"></location><text>URA3</text></annotation><annotation id="Team286_A880"><infon key="type">Gene</infon><location offset="59647" length="23"></location><text>ade2-101 ade3-24 leu2-3</text></annotation><annotation id="Team286_A881"><infon key="type">Gene</infon><location offset="59671" length="3"></location><text>112</text></annotation><annotation id="Team286_A882"><infon key="type">Gene</infon><location offset="59675" length="145"></location><text>ura3-52 CKY443 MATa prototroph Kaiser Lab Collection CKY473 MATaleu2-3,112 ura3-52 Gal+ Kaiser Lab Collection CKY534 MATalpha lst1Delta</text></annotation><annotation id="Team286_A883"><infon key="type">Gene</infon><location offset="59822" length="58"></location><text>LEU2 leu2-3,112 ura3-52 [pKR17HA] CKY535 MATalst1Delta</text></annotation><annotation id="Team286_A884"><infon key="type">Gene</infon><location offset="59882" length="58"></location><text>LEU2 leu2-3,112 ura3-52 [pKR17HA] CKY536 MATalst1Delta</text></annotation><annotation id="Team286_A885"><infon key="type">Gene</infon><location offset="59942" length="129"></location><text>LEU2 ura3-52 leu2-3,112 CKY540 MATaleu2-3,112 ura3-52 [pNV31] CKY541 MATasec12-4 ura3-52 [pNV31] CKY542 MATalst1Delta</text></annotation><annotation id="Team286_A886"><infon key="type">Gene</infon><location offset="60073" length="61"></location><text>LEU2 leu2-3,112 ura3-52 [pNV31] CKY552 MATalpha lst1Delta</text></annotation><annotation id="Team286_A887"><infon key="type">Gene</infon><location offset="60136" length="11"></location><text>LEU2 leu2-3</text></annotation><annotation id="Team286_A888"><infon key="type">Gene</infon><location offset="60148" length="3"></location><text>112</text></annotation><annotation id="Team286_A889"><infon key="type">Gene</infon><location offset="60152" length="7"></location><text>ura3-52</text></annotation></sentence></passage><passage><infon key="type">table_footnote</infon><offset>60114</offset><sentence><offset>60114</offset><text>All strains are from this study unless otherwise indicated.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>60180</offset><sentence><offset>60180</offset><text>Colony-sectoring screen for mutations that are lethal with sec13-1.</text><annotation id="Team286_A890"><infon key="type">Gene</infon><location offset="60240" length="7"></location><text>sec13-1</text></annotation></sentence><sentence><offset>60249</offset><text>CKY423 (ade2 ade3 leu2 ura3 sec13-1 [pKR4: SEC13, ADE3]) can lose the plasmid pKR4 when grown at 24 C on YPD, to give ade2 ade3 segregants that form white sectors within a red colony.</text><annotation id="Team286_A891"><infon key="type">Gene</infon><location offset="60249" length="6"></location><text>CKY423</text></annotation><annotation id="Team286_A892"><infon key="type">Gene</infon><location offset="60257" length="27"></location><text>ade2 ade3 leu2 ura3 sec13-1</text></annotation><annotation id="Team286_A893"><infon key="type">Gene</infon><location offset="60286" length="4"></location><text>pKR4</text></annotation><annotation id="Team286_A894"><infon key="type">Gene</infon><location offset="60293" length="5"></location><text>SEC13</text></annotation><annotation id="Team286_A895"><infon key="type">Gene</infon><location offset="60300" length="4"></location><text>ADE3</text></annotation><annotation id="Team286_A896"><infon key="type">Gene</infon><location offset="60369" length="20"></location><text>ade2 ade3 segregants</text></annotation></sentence><sentence><offset>60436</offset><text>Mutagenized cells that have acquired an lst mutation cannot grow without the pKR4 plasmid and form nonsectoring, solid red colonies.</text></sentence><sentence><offset>60570</offset><text>Of 132 nonsectoring colonies, the sectoring in 57 was restored by transformation with a second SEC13-bearing plasmid (pKR1).</text><annotation id="Team286_A897"><infon key="type">Gene</infon><location offset="60667" length="21"></location><text>SEC13-bearing plasmid</text></annotation><annotation id="Team286_A898"><infon key="type">Gene</infon><location offset="60690" length="4"></location><text>pKR1</text></annotation></sentence></passage><passage><infon key="type">table_caption</infon><offset>60698</offset><sentence><offset>60698</offset><text>Mutations Lethal with sec13-1</text><annotation id="Team286_A899"><infon key="type">Gene</infon><location offset="60720" length="7"></location><text>sec13-1</text></annotation></sentence></passage><passage><infon key="type">table</infon><offset>60729</offset><sentence><offset>60732</offset><text>Gene Number of alleles LST1 11 LST2 6 LST3 4 LST4 5 LST5 5 LST6 1 LST7 1 LST8 1 LST9 1 LST10 (SEC16) 2</text><annotation id="Team286_A900"><infon key="type">Gene</infon><location offset="60758" length="14"></location><text>LST1 11 LST2</text></annotation><annotation id="Team286_A901"><infon key="type">Gene</infon><location offset="60778" length="75"></location><text>LST3 4 LST4 5 LST5 5 LST6 1 LST7 1 LST8 1 LST9 1 LST10</text></annotation><annotation id="Team286_A902"><infon key="type">Gene</infon><location offset="60855" length="5"></location><text>SEC16</text></annotation></sentence></passage><passage><infon key="type">table_caption</infon><offset>60875</offset><sentence><offset>60875</offset><text>Growth of lst sec Double Mutants at 24 C</text></sentence></passage><passage><infon key="type">table</infon><offset>60918</offset><sentence><offset>60923</offset><text>sec13-1 sec16-2 sec23-1 sec31-1 sec17-1 sec18-1 lst1-1 - - - - + + lst2-1 - + + + ND ND lst3-1 - + + + ND ND lst4-1 +/- + + + ND ND lst5-1 - + + + ND ND lst6-1 - +/- - + - + + lst7-1 - + + + ND ND lst8-1 - + + + ND ND</text><annotation id="Team286_A903"><infon key="type">Gene</infon><location offset="60923" length="76"></location><text>sec13-1 sec16-2 sec23-1 sec31-1 sec17-1 sec18-1 lst1-1 - - - - +</text></annotation><annotation id="Team286_A904"><infon key="type">Gene</infon><location offset="61004" length="22"></location><text>lst2-1 - + + + ND</text></annotation><annotation id="Team286_A905"><infon key="type">Gene</infon><location offset="61032" length="52"></location><text>lst3-1 - + + + ND ND lst4-1 +/- + + + ND</text></annotation><annotation id="Team286_A906"><infon key="type">Gene</infon><location offset="61090" length="53"></location><text>lst5-1 - + + + ND ND lst6-1 - +/- - + - +</text></annotation><annotation id="Team286_A907"><infon key="type">Gene</infon><location offset="61148" length="54"></location><text>lst7-1 - + + + ND ND lst8-1 - + + + ND ND</text></annotation></sentence></passage><passage><infon key="type">table_footnote</infon><offset>61231</offset><sentence><offset>61231</offset><text>Growth is represented in decreasing order by: + &gt; +/- &gt; +/-- &gt; -.</text></sentence><sentence><offset>61297</offset><text>ND, not determined.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>61339</offset><sentence><offset>61339</offset><text>Comparison of LST1 and SEC24 sequences.</text><annotation id="Team286_A908"><infon key="type">Gene</infon><location offset="61353" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A909"><infon key="type">Gene</infon><location offset="61363" length="5"></location><text>SEC24</text></annotation></sentence><sentence><offset>61380</offset><text>Identities are indicated by solid lines and similarities are indicated by dotted lines.</text></sentence><sentence><offset>61470</offset><text>Overall amino acid identity is 23%.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>61507</offset><sentence><offset>61507</offset><text>Functional relationships between LST1 and SEC24.</text><annotation id="Team286_A910"><infon key="type">Gene</infon><location offset="61540" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A911"><infon key="type">Gene</infon><location offset="61549" length="5"></location><text>SEC24</text></annotation></sentence><sentence><offset>61557</offset><text>(A) Sensitivity of lst1Delta mutants to acidic medium.</text><annotation id="Team286_A912"><infon key="type">Gene</infon><location offset="61576" length="17"></location><text>lst1Delta mutants</text></annotation></sentence><sentence><offset>61612</offset><text>Equal numbers of wild-type (CKY443) or lst1Delta::LEU2 (CKY534) cells were spotted onto YPD medium, pH 6.5, or acidic YPD medium (brought to pH 3.8 by the addition of HCl).</text><annotation id="Team286_A913"><infon key="type">Gene</infon><location offset="61662" length="4"></location><text>LEU2</text></annotation><annotation id="Team286_A914"><infon key="type">Gene</infon><location offset="61668" length="6"></location><text>CKY534</text></annotation></sentence><sentence><offset>61787</offset><text>Plates were photographed after incubation at 37 C for 2 d.</text></sentence><sentence><offset>61846</offset><text>(B) A lst1Delta::LEU2 strain (CKY552) was transformed with: vector only, pRS316; LST1 on a centromeric plasmid, pKR17; SEC24 on a centromeric plasmid, pAF70; or SEC24 on a 2mu plasmid, pKR34; and streaked onto YPD medium, pH 3.8.</text><annotation id="Team286_A915"><infon key="type">Gene</infon><location offset="61863" length="4"></location><text>LEU2</text></annotation><annotation id="Team286_A916"><infon key="type">Gene</infon><location offset="61920" length="6"></location><text>pRS316</text></annotation><annotation id="Team286_A917"><infon key="type">Gene</infon><location offset="61929" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A918"><infon key="type">Gene</infon><location offset="61960" length="5"></location><text>pKR17</text></annotation><annotation id="Team286_A919"><infon key="type">Gene</infon><location offset="61967" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A920"><infon key="type">Gene</infon><location offset="62000" length="5"></location><text>pAF70</text></annotation><annotation id="Team286_A921"><infon key="type">Gene</infon><location offset="62010" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A922"><infon key="type">Gene</infon><location offset="62034" length="5"></location><text>pKR34</text></annotation></sentence><sentence><offset>62080</offset><text>Colonies were photographed after growth at 37 C for 2 d.</text></sentence><sentence><offset>62137</offset><text>(C) A wild-type strain (CKY473) was transformed either with a plasmid carrying pGAL1-LST1 (pKR35) and vector control (pRS425), or with pKR35 and SEC24 on a 2mu plasmid (pKR41).</text><annotation id="Team286_A923"><infon key="type">Gene</infon><location offset="62218" length="5"></location><text>pGAL1</text></annotation><annotation id="Team286_A924"><infon key="type">Gene</infon><location offset="62224" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A925"><infon key="type">Gene</infon><location offset="62230" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A926"><infon key="type">Gene</infon><location offset="62275" length="5"></location><text>pKR35</text></annotation><annotation id="Team286_A927"><infon key="type">Gene</infon><location offset="62285" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A928"><infon key="type">Gene</infon><location offset="62309" length="5"></location><text>pKR41</text></annotation></sentence><sentence><offset>62317</offset><text>Transformants were plated at a density of 800 cells/cm2 on SMM plates containing 2% raffinose and then 3 mg galactose solution was placed on a sterile 1-cm filter on top of the lawn.</text></sentence><sentence><offset>62502</offset><text>The plates were photographed after growth at 30 C for 2 d.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>62557</offset><sentence><offset>62557</offset><text>Pma1p accumulates the ER in lst1Delta cells and this accumulation is suppressed by overexpression of SEC24.</text><annotation id="Team286_A929"><infon key="type">Gene</infon><location offset="62557" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A930"><infon key="type">Gene</infon><location offset="62660" length="5"></location><text>SEC24</text></annotation></sentence><sentence><offset>62668</offset><text>Cells grown in SMM at 30 C were fixed with formaldehyde and then stained for immunofluorescence microscopy with affinity-purified anti-Pma1p antibody and FITC-conjugated secondary antibody.</text><annotation id="Team286_A931"><infon key="type">Gene</infon><location offset="62801" length="19"></location><text>anti-Pma1p antibody</text></annotation></sentence><sentence><offset>62862</offset><text>The same fields of cells, stained with DAPI to label the nuclear DNA, are also shown.</text></sentence><sentence><offset>62950</offset><text>Top panels, montage of lst1Delta cells (CKY536 carrying the empty vector pRS316); middle panels, genotypically wild-type cells (CKY536 carrying the LST1 plasmid pKR17); bottom panels, lst1Delta cells suppressed by SEC24 (CKY536 carrying the 2mu SEC24 plasmid pKR34).</text><annotation id="Team286_A932"><infon key="type">Gene</infon><location offset="62990" length="6"></location><text>CKY536</text></annotation><annotation id="Team286_A933"><infon key="type">Gene</infon><location offset="63101" length="18"></location><text>LST1 plasmid pKR17</text></annotation><annotation id="Team286_A934"><infon key="type">Gene</infon><location offset="63168" length="5"></location><text>SEC24</text></annotation><annotation id="Team286_A935"><infon key="type">Gene</infon><location offset="63175" length="6"></location><text>CKY536</text></annotation><annotation id="Team286_A936"><infon key="type">Gene</infon><location offset="63195" length="24"></location><text>2mu SEC24 plasmid pKR34</text></annotation></sentence><sentence><offset>63222</offset><text>Bar, 5 mum.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>63226</offset><sentence><offset>63226</offset><text>Pma1p defects caused by lst1Delta.</text><annotation id="Team286_A937"><infon key="type">Gene</infon><location offset="63226" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>63262</offset><text>(A) lst1Delta cells (CKY534) were photographed using differential interference contrast microscopy after growth at 37 C on YPD, pH 3.8.</text><annotation id="Team286_A938"><infon key="type">Gene</infon><location offset="63284" length="6"></location><text>CKY534</text></annotation></sentence><sentence><offset>63400</offset><text>A montage of multibudded cells is shown.</text></sentence><sentence><offset>63443</offset><text>Cells of this type comprise ~10% of a lst1Delta culture, but are never seen in wild-type grown under the same conditions.</text></sentence><sentence><offset>63568</offset><text>Bar, 10 mum.</text></sentence><sentence><offset>63581</offset><text>(B) Reduced capacity for proton pumping by lst1Delta cells.</text></sentence><sentence><offset>63642</offset><text>Wild-type (CKY443) and lst1Delta (CKY536) were grown to exponential phase in YPD medium, pH 6.8, at 37 C.</text><annotation id="Team286_A939"><infon key="type">Gene</infon><location offset="63665" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A940"><infon key="type">Gene</infon><location offset="63677" length="6"></location><text>CKY536</text></annotation></sentence><sentence><offset>63752</offset><text>Cells were incubated in water overnight and then suspended in 10 mM glycine buffer at pH 4.0.</text></sentence><sentence><offset>63847</offset><text>Proton efflux from the cells after addition of glucose was recorded as a decrease in the pH of the external medium.</text></sentence><sentence><offset>63963</offset><text>Based on the average rate of change in pH over the first 5 min after glucose addition, lst1Delta cells exhibited 65% the rate of proton efflux as wild-type.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>64108</offset><sentence><offset>64108</offset><text>Cell fraction to localize Pma1p in lst1Delta cells.</text><annotation id="Team286_A941"><infon key="type">Gene</infon><location offset="64134" length="5"></location><text>Pma1p</text></annotation></sentence><sentence><offset>64160</offset><text>Wild-type (CKY443) and lst1Delta (CKY536) cells were grown in YPD at 24 C and then were shifted to 37 C for 3 h.</text><annotation id="Team286_A942"><infon key="type">Gene</infon><location offset="64184" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A943"><infon key="type">Gene</infon><location offset="64195" length="6"></location><text>CKY536</text></annotation></sentence><sentence><offset>64275</offset><text>Cell lysates were fractionated on density gradients of 20-60% sucrose.</text></sentence><sentence><offset>64346</offset><text>Relative levels of Pma1p, Gas1p (plasma membrane marker), and Sec61p (ER marker) in each fraction were quantitated by immunoblotting and densitometry.</text><annotation id="Team286_A944"><infon key="type">Gene</infon><location offset="64366" length="5"></location><text>Pma1p</text></annotation><annotation id="Team286_A945"><infon key="type">Gene</infon><location offset="64373" length="5"></location><text>Gas1p</text></annotation><annotation id="Team286_A946"><infon key="type">Gene</infon><location offset="64409" length="6"></location><text>Sec61p</text></annotation><annotation id="Team286_A947"><infon key="type">Gene</infon><location offset="64417" length="2"></location><text>ER</text></annotation></sentence><sentence><offset>64500</offset><text>GDPase (Golgi compartment marker) was determined by enzymatic assay.</text><annotation id="Team286_A948"><infon key="type">Gene</infon><location offset="64500" length="6"></location><text>GDPase</text></annotation></sentence></passage><passage><infon key="type">fig_caption</infon><offset>64567</offset><sentence><offset>64567</offset><text>Transport of invertase is not affected by lst1Delta.</text><annotation id="Team286_A949"><infon key="type">Gene</infon><location offset="64580" length="9"></location><text>invertase</text></annotation></sentence><sentence><offset>64620</offset><text>Wild-type (CKY540), lst1Delta (CKY542), and sec12-4 (CKY541) strains expressing invertase from the constitutive pTPI1-SUC2 fusion, were grown to exponential phase at 24 C in SMM medium, pH 6.5, without methionine.</text><annotation id="Team286_A950"><infon key="type">Gene</infon><location offset="64620" length="18"></location><text>Wild-type (CKY540)</text></annotation><annotation id="Team286_A951"><infon key="type">Gene</infon><location offset="64640" length="9"></location><text>lst1Delta</text></annotation><annotation id="Team286_A952"><infon key="type">Gene</infon><location offset="64651" length="6"></location><text>CKY542</text></annotation><annotation id="Team286_A953"><infon key="type">Gene</infon><location offset="64664" length="7"></location><text>sec12-4</text></annotation><annotation id="Team286_A954"><infon key="type">Gene</infon><location offset="64673" length="6"></location><text>CKY541</text></annotation><annotation id="Team286_A955"><infon key="type">Gene</infon><location offset="64701" length="9"></location><text>invertase</text></annotation><annotation id="Team286_A956"><infon key="type">Gene</infon><location offset="64733" length="10"></location><text>pTPI1-SUC2</text></annotation></sentence><sentence><offset>64837</offset><text>Wild-type and lst1Delta strains were shifted to 37 C, grown for 3 h, and the sec12-4 (CKY541) strain was shifted to 37 C 5 min before labeling.</text><annotation id="Team286_A957"><infon key="type">Gene</infon><location offset="64915" length="7"></location><text>sec12-4</text></annotation><annotation id="Team286_A958"><infon key="type">Gene</infon><location offset="64924" length="6"></location><text>CKY541</text></annotation></sentence><sentence><offset>64983</offset><text>Cells were pulse-labeled with [35S]methionine and cysteine for 5 min and then chased by the addition of an excess of unlabeled methionine and cysteine.</text></sentence><sentence><offset>65136</offset><text>Invertase was immunoprecipitated from labeled extracts and resolved by SDS-PAGE.</text><annotation id="Team286_A959"><infon key="type">Gene</infon><location offset="65136" length="9"></location><text>Invertase</text></annotation></sentence><sentence><offset>65219</offset><text>Positions of the core glycosylated ER form and mature Golgi and secreted forms of invertase are indicated.</text><annotation id="Team286_A960"><infon key="type">Gene</infon><location offset="65301" length="9"></location><text>invertase</text></annotation></sentence></passage><passage><infon key="type">fig_caption</infon><offset>65322</offset><sentence><offset>65322</offset><text>Immunolocalization of Lst1p-HA.</text><annotation id="Team286_A961"><infon key="type">Gene</infon><location offset="65344" length="8"></location><text>Lst1p-HA</text></annotation></sentence><sentence><offset>65354</offset><text>CKY535 (MATa lst1Delta::LEU2 leu2-3,112 ura3-52 [pKR17HA]) expressing Lst1p-HA from a centromeric plasmid was fixed and labeled with mouse anti-HA, FITC-conjugated anti-mouse antibodies, rabbit anti-Kar2p, and rhodamine-conjugated anti-rabbit antibodies.</text><annotation id="Team286_A962"><infon key="type">Gene</infon><location offset="65354" length="6"></location><text>CKY535</text></annotation><annotation id="Team286_A963"><infon key="type">Gene</infon><location offset="65362" length="15"></location><text>MATa lst1Delta</text></annotation><annotation id="Team286_A964"><infon key="type">Gene</infon><location offset="65379" length="23"></location><text>LEU2 leu2-3,112 ura3-52</text></annotation><annotation id="Team286_A965"><infon key="type">Gene</infon><location offset="65404" length="7"></location><text>pKR17HA</text></annotation><annotation id="Team286_A966"><infon key="type">Gene</infon><location offset="65425" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A967"><infon key="type">Gene</infon><location offset="65489" length="13"></location><text>mouse anti-HA</text></annotation><annotation id="Team286_A968"><infon key="type">Gene</infon><location offset="65504" length="37"></location><text>FITC-conjugated anti-mouse antibodies</text></annotation><annotation id="Team286_A969"><infon key="type">Gene</infon><location offset="65543" length="18"></location><text>rabbit anti-Kar2p</text></annotation><annotation id="Team286_A970"><infon key="type">Gene</infon><location offset="65588" length="22"></location><text>anti-rabbit antibodies</text></annotation></sentence><sentence><offset>65613</offset><text>Nuclear DNA was visualized by DAPI staining.</text></sentence><sentence><offset>65658</offset><text>Cell bodies were visualized by differential interference contrast microscopy (DIC).</text></sentence><sentence><offset>65744</offset><text>Bar, 1 mum.</text></sentence></passage><passage><infon key="type">fig_caption</infon><offset>65757</offset><sentence><offset>65757</offset><text>The intracellular distribution of Lst1p.</text><annotation id="Team286_A971"><infon key="type">Gene</infon><location offset="65791" length="5"></location><text>Lst1p</text></annotation></sentence><sentence><offset>65798</offset><text>(A) Cells expressing Lst1p-HA from a centromeric plasmid (CKY535) were gently lysed and subjected to sequential centrifugation steps, giving 500 g, 10,000 g, and 150,000 g pellet fractions (P) and a 150,000 g supernatant fraction (S).</text><annotation id="Team286_A972"><infon key="type">Gene</infon><location offset="65819" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A973"><infon key="type">Gene</infon><location offset="65856" length="6"></location><text>CKY535</text></annotation></sentence><sentence><offset>66035</offset><text>Each sample contains extract from the same number of cells.</text></sentence><sentence><offset>66096</offset><text>(B) Cell lysates were treated for 1 h at 4 C with either 2.5 M urea, 500 mM NaCl, 100 mM sodium carbonate (pH 11.5), or 1% Triton X-100.</text></sentence><sentence><offset>66234</offset><text>Pellet (P) and supernatant (S) fractions were then separated by centrifugation at 50,000 g.</text></sentence><sentence><offset>66328</offset><text>Lst1p-HA was detected by SDS-PAGE and immunoblotting with anti-HA antibody.</text><annotation id="Team286_A974"><infon key="type">Gene</infon><location offset="66328" length="8"></location><text>Lst1p-HA</text></annotation><annotation id="Team286_A975"><infon key="type">Gene</infon><location offset="66387" length="16"></location><text>anti-HA antibody</text></annotation></sentence></passage><passage><infon key="type">table_caption</infon><offset>66406</offset><sentence><offset>66406</offset><text>Two-Hybrid Interaction between LST1 and SEC23</text><annotation id="Team286_A976"><infon key="type">Gene</infon><location offset="66437" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A977"><infon key="type">Gene</infon><location offset="66446" length="5"></location><text>SEC23</text></annotation></sentence></passage><passage><infon key="type">table</infon><offset>66453</offset><sentence><offset>66458</offset><text>beta-galactosidase activity LST1 SEC24 No fusion SEC23 395 +- 8 629 +- 1 24.4 +- 0.1 No fusion 30.4 +- 4.0 25.3 +- 1.4 44.8 +- 5.3</text><annotation id="Team286_A978"><infon key="type">Gene</infon><location offset="66458" length="18"></location><text>beta-galactosidase</text></annotation><annotation id="Team286_A979"><infon key="type">Gene</infon><location offset="66487" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A980"><infon key="type">Gene</infon><location offset="66493" length="5"></location><text>SEC24</text></annotation></sentence></passage><passage><infon key="type">table_footnote</infon><offset>66599</offset><sentence><offset>66599</offset><text>Fusions to the LexA DNA-binding domain and to a transcriptional activation domain were induced by growth in galactose for 10 h.</text><annotation id="Team286_A981"><infon key="type">Gene</infon><location offset="66614" length="23"></location><text>LexA DNA-binding domain</text></annotation></sentence><sentence><offset>66728</offset><text>Activities shown are the mean from five independent transformants.</text></sentence><sentence><offset>66796</offset><text>Units of beta-galactosidase activity are nmol/mg x min.</text><annotation id="Team286_A982"><infon key="type">Gene</infon><location offset="66805" length="18"></location><text>beta-galactosidase</text></annotation></sentence></passage><passage><infon key="type">fig_caption</infon><offset>66859</offset><sentence><offset>66859</offset><text>Lst1p/-Sec23p complex is membrane associated.</text><annotation id="Team286_A983"><infon key="type">Gene</infon><location offset="66859" length="21"></location><text>Lst1p/-Sec23p complex</text></annotation></sentence><sentence><offset>66905</offset><text>(A) Affinity isolation of Lst1p/-Sec23p complexes.</text><annotation id="Team286_A984"><infon key="type">Gene</infon><location offset="66932" length="23"></location><text>Lst1p/-Sec23p complexes</text></annotation></sentence><sentence><offset>66957</offset><text>GST-Lst1p-HA or GST alone was coexpressed with Sec23p and isolated by affinity to glutathione Sepharose beads.</text><annotation id="Team286_A985"><infon key="type">Gene</infon><location offset="66957" length="3"></location><text>GST</text></annotation><annotation id="Team286_A986"><infon key="type">Gene</infon><location offset="66961" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A987"><infon key="type">Gene</infon><location offset="66967" length="2"></location><text>HA</text></annotation><annotation id="Team286_A988"><infon key="type">Gene</infon><location offset="66974" length="3"></location><text>GST</text></annotation><annotation id="Team286_A989"><infon key="type">Gene</infon><location offset="67005" length="6"></location><text>Sec23p</text></annotation></sentence><sentence><offset>67069</offset><text>Proteins bound to glutathione beads were loaded in lanes 2 and 4.</text></sentence><sentence><offset>67135</offset><text>One-sixth of the total lysate was loaded in lanes 1 and 3.</text></sentence><sentence><offset>67194</offset><text>(B) GST-LST1-HA, SEC23, or both were expressed from the GAL1 promoter.</text><annotation id="Team286_A990"><infon key="type">Gene</infon><location offset="67198" length="3"></location><text>GST</text></annotation><annotation id="Team286_A991"><infon key="type">Gene</infon><location offset="67202" length="4"></location><text>LST1</text></annotation><annotation id="Team286_A992"><infon key="type">Gene</infon><location offset="67207" length="2"></location><text>HA</text></annotation><annotation id="Team286_A993"><infon key="type">Gene</infon><location offset="67212" length="5"></location><text>SEC23</text></annotation><annotation id="Team286_A994"><infon key="type">Gene</infon><location offset="67251" length="13"></location><text>GAL1 promoter</text></annotation></sentence><sentence><offset>67266</offset><text>Cell lysates were cleared of cell debris by centrifugation at 300 g for 2 min.</text></sentence><sentence><offset>67347</offset><text>Pellet (P) and supernatant (S) fractions from cleared cell lysates were separated by centrifugation at 10,000 g for 30 min.</text></sentence><sentence><offset>67471</offset><text>An aliquot of the total cleared lysate (T) was removed before centrifugation.</text></sentence><sentence><offset>67550</offset><text>An equal number of cell equivalents were loaded for each sample.</text></sentence><sentence><offset>67616</offset><text>The GST-Lst1p-HA fusion was detected using anti-HA antibodies.</text><annotation id="Team286_A995"><infon key="type">Gene</infon><location offset="67620" length="3"></location><text>GST</text></annotation><annotation id="Team286_A996"><infon key="type">Gene</infon><location offset="67624" length="5"></location><text>Lst1p</text></annotation><annotation id="Team286_A997"><infon key="type">Gene</infon><location offset="67630" length="2"></location><text>HA</text></annotation><annotation id="Team286_A998"><infon key="type">Gene</infon><location offset="67659" length="18"></location><text>anti-HA antibodies</text></annotation></sentence><sentence><offset>67679</offset><text>For both A and B, the Sec23p protein was detected using anti-Sec23p antibodies.</text><annotation id="Team286_A999"><infon key="type">Gene</infon><location offset="67701" length="14"></location><text>Sec23p protein</text></annotation><annotation id="Team286_A1000"><infon key="type">Gene</infon><location offset="67735" length="22"></location><text>anti-Sec23p antibodies</text></annotation></sentence></passage><passage><infon key="type">title</infon><offset>67769</offset><sentence><offset>67769</offset><text>Abbreviations used in this paper</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67802</offset><sentence><offset>67802</offset><text>DAPI</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67807</offset><sentence><offset>67807</offset><text>4',6-diamidino-2-phenylindole</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67839</offset><sentence><offset>67839</offset><text>GST</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67843</offset><sentence><offset>67843</offset><text>glutathione S-transferase</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67869</offset><sentence><offset>67869</offset><text>HA</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67872</offset><sentence><offset>67872</offset><text>hemagglutinin epitope</text><annotation id="Team286_A1001"><infon key="type">Gene</infon><location offset="67872" length="13"></location><text>hemagglutinin</text></annotation></sentence></passage><passage><infon key="type">paragraph</infon><offset>67894</offset><sentence><offset>67894</offset><text>LST</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67898</offset><sentence><offset>67898</offset><text>lethal with sec-thirteen</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67924</offset><sentence><offset>67924</offset><text>Pma1p</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67930</offset><sentence><offset>67930</offset><text>plasma membrane proton-ATPase</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67960</offset><sentence><offset>67960</offset><text>SMM</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67964</offset><sentence><offset>67964</offset><text>supplemented minimal medium</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67992</offset><sentence><offset>67992</offset><text>YPD</text></sentence></passage><passage><infon key="type">paragraph</infon><offset>67996</offset><sentence><offset>67996</offset><text>rich medium</text></sentence></passage><passage><infon key="type">title</infon><offset>68008</offset><sentence><offset>68008</offset><text>References</text></sentence></passage><passage><infon key="source">Proc Natl Acad Sci USA</infon><infon key="name_3">surname:Hirschberg;given-names:CB</infon><infon key="name_2">surname:Robbins;given-names:PW</infon><infon key="name_1">surname:Orlean;given-names:P</infon><infon key="pub-id_pmid">2476806</infon><infon key="name_0">surname:Abeijon;given-names:C</infon><infon key="volume">86</infon><infon key="year">1989</infon><infon key="type">ref</infon><infon key="fpage">6935</infon><infon key="lpage">6939</infon><offset>68019</offset><text>Topography of glycosylation in yeast: characterization of GDP mannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_3">surname:Nuoffer;given-names:C</infon><infon key="name_4">surname:Balch;given-names:WE</infon><infon key="name_2">surname:Bannykh;given-names:S</infon><infon key="name_1">surname:Weissman;given-names:J</infon><infon key="pub-id_pmid">9531548</infon><infon key="name_0">surname:Aridor;given-names:M</infon><infon key="volume">141</infon><infon key="year">1998</infon><infon key="type">ref</infon><infon key="fpage">61</infon><infon key="lpage">70</infon><offset>68169</offset><text>Cargo selection by the COPII budding machinery during export from the ER</text></passage><passage><infon key="source">Nature</infon><infon key="name_1">surname:Schekman;given-names:R</infon><infon key="pub-id_pmid">8377826</infon><infon key="name_0">surname:Barlowe;given-names:C</infon><infon key="volume">365</infon><infon key="year">1993</infon><infon key="type">ref</infon><infon key="fpage">347</infon><infon key="lpage">349</infon><offset>68242</offset><text>SEC12encodes a guanine-nucleotide- exchange factor essential for transport vesicle budding from the ER</text></passage><passage><infon key="name_9">surname:Schekman;given-names:R</infon><infon key="name_8">surname:Amherdt;given-names:M</infon><infon key="name_5">surname:Salama;given-names:N</infon><infon key="name_4">surname:Hamamoto;given-names:S</infon><infon key="name_7">surname:Ravazzola;given-names:M</infon><infon key="pub-id_pmid">8004676</infon><infon key="name_6">surname:Rexach;given-names:MF</infon><infon key="type">ref</infon><infon key="lpage">907</infon><infon key="source">Cell</infon><infon key="name_3">surname:Hosobuchi;given-names:M</infon><infon key="name_2">surname:Yeung;given-names:T</infon><infon key="name_1">surname:Orci;given-names:L</infon><infon key="name_0">surname:Barlowe;given-names:C</infon><infon key="volume">77</infon><infon key="year">1994</infon><infon key="fpage">895</infon><offset>68345</offset><text>COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum</text></passage><passage><infon key="source">Methods Enzymol</infon><infon key="name_1">surname:Fields;given-names:S</infon><infon key="pub-id_pmid">8531690</infon><infon key="name_0">surname:Bartel;given-names:PL</infon><infon key="volume">254</infon><infon key="year">1995</infon><infon key="type">ref</infon><infon key="fpage">241</infon><infon key="lpage">263</infon><offset>68449</offset><text>Analyzing protein-protein interactions using two-hybrid system</text></passage><passage><infon key="source">J Bacteriol</infon><infon key="name_1">surname:Schekman;given-names:R</infon><infon key="pub-id_pmid">2967284</infon><infon key="name_0">surname:Brada;given-names:D</infon><infon key="volume">170</infon><infon key="year">1988</infon><infon key="type">ref</infon><infon key="fpage">2775</infon><infon key="lpage">2783</infon><offset>68514</offset><text>Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_1">surname:Slayman;given-names:CW</infon><infon key="pub-id_pmid">1833410</infon><infon key="name_0">surname:Chang;given-names:A</infon><infon key="volume">115</infon><infon key="year">1991</infon><infon key="type">ref</infon><infon key="fpage">289</infon><infon key="lpage">295</infon><offset>68625</offset><text>Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_1">surname:Fink;given-names:GR</infon><infon key="pub-id_pmid">7822420</infon><infon key="name_0">surname:Chang;given-names:A</infon><infon key="volume">128</infon><infon key="year">1995</infon><infon key="type">ref</infon><infon key="fpage">39</infon><infon key="lpage">49</infon><offset>68733</offset><text>Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1prevents mislocalization of mutant ATPase to the vacuole</text></passage><passage><infon key="citation">Cherry, J.M., C. Adler, C. Ball, S. Dwight, S. Chervitz, Y. Jia, G. Juvik, S. Weng, and D. Botstein. 1997. Saccharomyces Genome Database. http:// genome-www.stanford.edu/Saccharomyces/</infon><infon key="type">ref</infon><offset>68859</offset></passage><passage><infon key="source">Curr Genet</infon><infon key="name_2">surname:Serrano;given-names:R</infon><infon key="name_1">surname:Perona;given-names:R</infon><infon key="pub-id_pmid">2966684</infon><infon key="name_0">surname:Cid;given-names:A</infon><infon key="volume">12</infon><infon key="year">1987</infon><infon key="type">ref</infon><infon key="fpage">105</infon><infon key="lpage">110</infon><offset>68860</offset><text>Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences</text></passage><passage><infon key="source">J Biol Chem</infon><infon key="pub-id_pmid">7559560</infon><infon key="volume">270</infon><infon key="year">1995</infon><infon key="type">ref</infon><infon key="fpage">23828</infon><infon key="lpage">23837</infon><offset>68998</offset><text>Functional complementation of a null mutation of the yeast Saccharomyces cerevisiaeplasma membrane H(+)-ATPase by a plant H(+)-ATPase gene</text></passage><passage><infon key="source">Proc Natl Acad Sci USA</infon><infon key="name_3">surname:Spottswood;given-names:M</infon><infon key="name_4">surname:Davis;given-names:RW</infon><infon key="name_2">surname:Ramer;given-names:SW</infon><infon key="name_1">surname:Mulligan;given-names:JT</infon><infon key="pub-id_pmid">1848010</infon><infon key="name_0">surname:Elledge;given-names:SJ</infon><infon key="volume">88</infon><infon key="year">1991</infon><infon key="type">ref</infon><infon key="fpage">1731</infon><infon key="lpage">1735</infon><offset>69137</offset><text>Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia colimutations</text></passage><passage><infon key="source">Mol Biol Cell</infon><infon key="name_1">surname:Kaiser;given-names:CA</infon><infon key="pub-id_pmid">8862519</infon><infon key="name_0">surname:Elrod-Erickson;given-names:MJ</infon><infon key="volume">7</infon><infon key="year">1996</infon><infon key="type">ref</infon><infon key="fpage">1043</infon><infon key="lpage">1058</infon><offset>69275</offset><text>Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_3">surname:Teung;given-names:P</infon><infon key="name_4">surname:Kaiser;given-names:CA</infon><infon key="name_2">surname:Holzmacher;given-names:E</infon><infon key="name_1">surname:Gimeno;given-names:RE</infon><infon key="pub-id_pmid">7593161</infon><infon key="name_0">surname:Espenshade;given-names:P</infon><infon key="volume">131</infon><infon key="year">1995</infon><infon key="type">ref</infon><infon key="fpage">311</infon><infon key="lpage">324</infon><offset>69406</offset><text>Yeast SEC16gene encodes a multidomain vesicle coat protein that interacts with Sec23p</text></passage><passage><infon key="citation">Gimeno, R.E. 1996. Proteins that interact with Sec16p during COPII vesicle formation in Saccharomyces cerevisiae. Ph.D. thesis. Massachusetts Institute of Technology, Cambridge, Massachusetts. 215 pp.</infon><infon key="type">ref</infon><offset>69492</offset></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_2">surname:Kaiser;given-names:CA</infon><infon key="name_1">surname:Espenshade;given-names:P</infon><infon key="pub-id_pmid">7593162</infon><infon key="name_0">surname:Gimeno;given-names:RE</infon><infon key="volume">131</infon><infon key="year">1995</infon><infon key="type">ref</infon><infon key="fpage">325</infon><infon key="lpage">338</infon><offset>69493</offset><text>SED4encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation</text></passage><passage><infon key="source">Mol Cell Biol</infon><infon key="name_2">surname:Kaiser;given-names:CA</infon><infon key="name_1">surname:Espenshade;given-names:P</infon><infon key="name_0">surname:Gimeno;given-names:RE</infon><infon key="volume">7</infon><infon key="year">1996</infon><infon key="type">ref</infon><infon key="fpage">1815</infon><infon key="lpage">1823</infon><offset>69599</offset><text>COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p</text></passage><passage><infon key="source">Mol Cell Biol</infon><infon key="name_1">surname:Brent;given-names:R</infon><infon key="pub-id_pmid">1620111</infon><infon key="name_0">surname:Golemis;given-names:EA</infon><infon key="volume">12</infon><infon key="year">1992</infon><infon key="type">ref</infon><infon key="fpage">3006</infon><infon key="lpage">3014</infon><offset>69685</offset><text>Fused protein domains inhibit DNA binding by LexA</text></passage><passage><infon key="source">Cell</infon><infon key="name_3">surname:Brent;given-names:R</infon><infon key="name_2">surname:Chertkov;given-names:H</infon><infon key="name_1">surname:Golemis;given-names:E</infon><infon key="pub-id_pmid">8242750</infon><infon key="name_0">surname:Gyuris;given-names:J</infon><infon key="volume">75</infon><infon key="year">1993</infon><infon key="type">ref</infon><infon key="fpage">791</infon><infon key="lpage">803</infon><offset>69735</offset><text>Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2</text></passage><passage><infon key="citation">Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.</infon><infon key="type">ref</infon><offset>69810</offset></passage><passage><infon key="source">J Biol Chem</infon><infon key="name_5">surname:Teem;given-names:JH</infon><infon key="name_3">surname:Seto;given-names:YD</infon><infon key="name_4">surname:Perlin;given-names:DS</infon><infon key="name_2">surname:Zhu;given-names:X</infon><infon key="name_1">surname:Na;given-names:S</infon><infon key="pub-id_pmid">1837022</infon><infon key="name_6">surname:Haber;given-names:JE</infon><infon key="name_0">surname:Harris;given-names:SL</infon><infon key="volume">266</infon><infon key="year">1994</infon><infon key="type">ref</infon><infon key="fpage">24439</infon><infon key="lpage">24445</infon><offset>69811</offset><text>Dominant lethal mutations in the plasma membrane H+-ATPase gene of Saccharomyces cerevisiae. </text></passage><passage><infon key="source">Methods Enzymol</infon><infon key="name_1">surname:Jensen;given-names:RE</infon><infon key="pub-id_pmid">2005783</infon><infon key="name_0">surname:Herskowitz;given-names:I</infon><infon key="volume">155</infon><infon key="year">1991</infon><infon key="type">ref</infon><infon key="fpage">132</infon><infon key="lpage">146</infon><offset>69906</offset><text>Putting the HO gene to work: practical uses for mating-type switching</text></passage><passage><infon key="source">Mol Biol Cell</infon><infon key="name_2">surname:Schekman;given-names:R</infon><infon key="name_1">surname:Yoshihisa;given-names:T</infon><infon key="pub-id_pmid">1498369</infon><infon key="name_0">surname:Hicke;given-names:L</infon><infon key="volume">3</infon><infon key="year">1992</infon><infon key="type">ref</infon><infon key="fpage">667</infon><infon key="lpage">676</infon><offset>69976</offset><text>Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum</text></passage><passage><infon key="source">Mol Biol Cell</infon><infon key="name_1">surname:Stevens;given-names:TH</infon><infon key="pub-id_pmid">7841520</infon><infon key="name_0">surname:Hill;given-names:KJ</infon><infon key="volume">5</infon><infon key="year">1994</infon><infon key="type">ref</infon><infon key="fpage">1039</infon><infon key="lpage">1050</infon><offset>70124</offset><text>Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H+-ATPase complex</text></passage><passage><infon key="source">J Biol Chem</infon><infon key="name_1">surname:Stevens;given-names:TH</infon><infon key="pub-id_pmid">7673216</infon><infon key="name_0">surname:Hill;given-names:KJ</infon><infon key="volume">270</infon><infon key="year">1995</infon><infon key="type">ref</infon><infon key="fpage">22329</infon><infon key="lpage">22336</infon><offset>70286</offset><text>Vma22p is a novel endoplasmic reticulum- associated protein required for assembly of the yeast vacuolar H+-ATPase complex</text></passage><passage><infon key="source">J Biol Chem</infon><infon key="name_1">surname:Stevens;given-names:TH</infon><infon key="pub-id_pmid">9325326</infon><infon key="name_0">surname:Jackson;given-names:DD</infon><infon key="volume">272</infon><infon key="year">1997</infon><infon key="type">ref</infon><infon key="fpage">25928</infon><infon key="lpage">25934</infon><offset>70408</offset><text>VMA12 encodes a yeast endoplasmic reticulum protein required for vacuolar H+-ATPase assembly</text></passage><passage><infon key="source">Yeast</infon><infon key="name_1">surname:Prakash;given-names:L</infon><infon key="pub-id_pmid">2220072</infon><infon key="name_0">surname:Jones;given-names:JS</infon><infon key="volume">6</infon><infon key="year">1990</infon><infon key="type">ref</infon><infon key="fpage">363</infon><infon key="lpage">366</infon><offset>70502</offset><text>Yeast Saccharomyces cerevisiaeselectable markers in pUC18 polylinkers</text></passage><passage><infon key="source">Cell</infon><infon key="name_1">surname:Schekman;given-names:R</infon><infon key="pub-id_pmid">2188733</infon><infon key="name_0">surname:Kaiser;given-names:CA</infon><infon key="volume">61</infon><infon key="year">1990</infon><infon key="type">ref</infon><infon key="fpage">723</infon><infon key="lpage">733</infon><offset>70572</offset><text>Distinct sets of SECgenes govern transport vesicle formation and fusion early in the secretory pathway</text></passage><passage><infon key="citation">Kaiser, C.A., S. Michaelis, and A. Mitchell. 1994. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.</infon><infon key="type">ref</infon><offset>70675</offset></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_2">surname:Ljungdahl;given-names:P</infon><infon key="name_1">surname:Schekman;given-names:R</infon><infon key="pub-id_pmid">8909535</infon><infon key="name_0">surname:Kuehn;given-names:MJ</infon><infon key="volume">135</infon><infon key="year">1996</infon><infon key="type">ref</infon><infon key="fpage">585</infon><infon key="lpage">595</infon><offset>70676</offset><text>Amino acid permeases require COPII components and the ER resident membrane-protein Shr3p for packaging into transport vesicles in vitro</text></passage><passage><infon key="source">Science</infon><infon key="name_2">surname:Schekman;given-names:R</infon><infon key="name_1">surname:Herrmann;given-names:JM</infon><infon key="name_0">surname:Kuehn;given-names:MJ</infon><infon key="volume">391</infon><infon key="year">1998</infon><infon key="type">ref</infon><infon key="fpage">187</infon><infon key="lpage">190</infon><offset>70812</offset><text>COPII-cargo interactions direct protein sorting into ER-derived transport vesicles</text></passage><passage><infon key="source">Methods Enzymol</infon><infon key="name_0">surname:Lawrence;given-names:CW</infon><infon key="volume">155</infon><infon key="year">1991</infon><infon key="type">ref</infon><infon key="fpage">132</infon><infon key="lpage">146</infon><offset>70895</offset><text>Classical mutagenesis techniques</text></passage><passage><infon key="source">Cell</infon><infon key="name_3">surname:Fink;given-names:GR</infon><infon key="name_2">surname:Styles;given-names:CA</infon><infon key="name_1">surname:Gimeno;given-names:CJ</infon><infon key="pub-id_pmid">1423607</infon><infon key="name_0">surname:Ljungdahl;given-names:PO</infon><infon key="volume">71</infon><infon key="year">1992</infon><infon key="type">ref</infon><infon key="fpage">463</infon><infon key="lpage">478</infon><offset>70928</offset><text>SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast</text></passage><passage><infon key="source">Cell</infon><infon key="name_5">surname:Schekman;given-names:R</infon><infon key="name_3">surname:Bednarek;given-names:SY</infon><infon key="name_4">surname:Hamamoto;given-names:S</infon><infon key="name_2">surname:Amherdt;given-names:M</infon><infon key="name_1">surname:Orci;given-names:L</infon><infon key="pub-id_pmid">9568718</infon><infon key="name_6">surname:Yeung;given-names:T</infon><infon key="name_0">surname:Matsuoka;given-names:K</infon><infon key="volume">93</infon><infon key="year">1998</infon><infon key="type">ref</infon><infon key="fpage">263</infon><infon key="lpage">275</infon><offset>71049</offset><text>COPII-coated vesicle formation reconstituted with purified coat proteins and chemically-defined liposomes</text></passage><passage><infon key="source">Mol Cell Biol</infon><infon key="name_2">surname:Haber;given-names:JE</infon><infon key="name_1">surname:Perlin;given-names:DS</infon><infon key="pub-id_pmid">2963211</infon><infon key="name_0">surname:McCusker;given-names:JH</infon><infon key="volume">7</infon><infon key="year">1987</infon><infon key="type">ref</infon><infon key="fpage">4082</infon><infon key="lpage">4088</infon><offset>71155</offset><text>Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae. </text></passage><passage><infon key="source">J Bacteriol</infon><infon key="name_1">surname:Magasanik;given-names:B</infon><infon key="pub-id_pmid">1975578</infon><infon key="name_0">surname:Miller;given-names:S</infon><infon key="volume">172</infon><infon key="year">1990</infon><infon key="type">ref</infon><infon key="fpage">4927</infon><infon key="lpage">4935</infon><offset>71230</offset><text>Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae. </text></passage><passage><infon key="source">Mol Cell Biol</infon><infon key="name_3">surname:Riezman;given-names:H</infon><infon key="name_2">surname:Conzelmann;given-names:A</infon><infon key="name_1">surname:Jeno;given-names:P</infon><infon key="pub-id_pmid">1824714</infon><infon key="name_0">surname:Nuoffer;given-names:C</infon><infon key="volume">11</infon><infon key="year">1991</infon><infon key="type">ref</infon><infon key="fpage">27</infon><infon key="lpage">37</infon><offset>71326</offset><text>Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiaeGAS1 protein to the plasma membrane</text></passage><passage><infon key="source">FEBS Lett</infon><infon key="pub-id_pmid">9037182</infon><infon key="name_0">surname:Portillo;given-names:F</infon><infon key="volume">402</infon><infon key="year">1997</infon><infon key="type">ref</infon><infon key="fpage">136</infon><infon key="lpage">140</infon><offset>71439</offset><text>Characterization of dominant lethal mutations in the yeast plasma-membrane H+-ATPase gene</text></passage><passage><infon key="source">Eur J Biochem</infon><infon key="name_1">surname:Serrano;given-names:R</infon><infon key="pub-id_pmid">2532597</infon><infon key="name_0">surname:Portillo;given-names:F</infon><infon key="volume">186</infon><infon key="year">1989</infon><infon key="type">ref</infon><infon key="fpage">501</infon><infon key="lpage">507</infon><offset>71530</offset><text>Growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_1">surname:Barlowe;given-names:C</infon><infon key="pub-id_pmid">9732282</infon><infon key="name_0">surname:Powers;given-names:J</infon><infon key="volume">142</infon><infon key="year">1998</infon><infon key="type">ref</infon><infon key="fpage">1209</infon><infon key="lpage">1222</infon><offset>71639</offset><text>Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophilacornichon gene product</text></passage><passage><infon key="source">Methods Enzymol</infon><infon key="name_3">surname:Haarer;given-names:BK</infon><infon key="name_2">surname:Drubin;given-names:DG</infon><infon key="name_1">surname:Adams;given-names:AEM</infon><infon key="pub-id_pmid">2005809</infon><infon key="name_0">surname:Pringle;given-names:JR</infon><infon key="volume">194</infon><infon key="year">1991</infon><infon key="type">ref</infon><infon key="fpage">565</infon><infon key="lpage">602</infon><offset>71748</offset><text>Immuno-fluorescence methods for yeast</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_3">surname:Kaiser;given-names:CA</infon><infon key="name_2">surname:Schekman;given-names:R</infon><infon key="name_1">surname:Salama;given-names:NR</infon><infon key="pub-id_pmid">8432727</infon><infon key="name_0">surname:Pryer;given-names:NK</infon><infon key="volume">120</infon><infon key="year">1993</infon><infon key="type">ref</infon><infon key="fpage">865</infon><infon key="lpage">875</infon><offset>71786</offset><text>Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro</text></passage><passage><infon key="source">J Cell Biol</infon><infon key="name_2">surname:Kaiser;given-names:CA</infon><infon key="name_1">surname:Rowley;given-names:N</infon><infon key="pub-id_pmid">9199164</infon><infon key="name_0">surname:Roberg;given-names:KJ</infon><infon key="volume">137</infon><infon key="year">1997a</infon><infon key="type">ref</infon><infon key="fpage">1469</infon><infon key="lpage">1482</infon><offset>71885</offset><text>Physiological regulation of membrane protein sorting in the Golgi of Saccharomyces cerevisiae. </text></passage><passage><infon key="source">Genetics</infon><infon key="name_3">surname:Kaiser;given-names:CA</infon><infon key="name_2">surname:Rowley;given-names:N</infon><infon key="name_1">surname:Bickel;given-names:S</infon><infon key="pub-id_pmid">9409822</infon><infon key="name_0">surname:Roberg;given-names:KJ</infon><infon key="volume">147</infon><infon key="year">1997b</infon><infon key="type">ref</infon><infon key="fpage">1569</infon><infon key="lpage">1584</infon><offset>71982</offset><text>Control of amino-acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7, and LST8. </text></passage><passage><infon key="source">Methods Enzymol</infon><infon key="name_1">surname:Botstein;given-names:D</infon><infon key="pub-id_pmid">6310320</infon><infon key="name_0">surname:Rose;given-names:M</infon><infon key="volume">101</infon><infon key="year">1983</infon><infon key="type">ref</infon><infon key="fpage">167</infon><infon key="lpage">180</infon><offset>72112</offset><text>Construction and use of gene fusions lacZ(beta-galactosidase) which are expressed in yeast</text></passage><passage><infon key="citation">Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. </infon><infon key="type">ref</infon><offset>72201</offset></passage><passage><infon key="source">Science</infon><infon key="name_1">surname:Orci;given-names:L</infon><infon key="pub-id_pmid">8599108</infon><infon key="name_0">surname:Schekman;given-names:R</infon><infon key="volume">271</infon><infon key="year">1996</infon><infon key="type">ref</infon><infon key="fpage">1526</infon><infon key="lpage">1533</infon><offset>72202</offset><text>Coat proteins and vesicle budding</text></passage><passage><infon key="citation">Serrano, R. 1991. Transport across yeast vacuolar and plasma membranes. In The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics. J.R. Broach, E.W. Jones, and J.R. Pringle, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 523-585.</infon><infon key="type">ref</infon><offset>72236</offset></passage><passage><infon key="source">Nature</infon><infon key="name_2">surname:Fink;given-names:GR</infon><infon key="name_1">surname:Kieland-Brandt;given-names:MC</infon><infon key="pub-id_pmid">3005867</infon><infon key="name_0">surname:Serrano;given-names:R</infon><infon key="volume">319</infon><infon key="year">1986</infon><infon key="type">ref</infon><infon key="fpage">689</infon><infon key="lpage">693</infon><offset>72237</offset><text>Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPase</text></passage><passage><infon key="source">J Biol Chem</infon><infon key="name_3">surname:Kaiser;given-names:CA</infon><infon key="name_2">surname:Gimeno;given-names:RE</infon><infon key="name_1">surname:Espenshade;given-names:PJ</infon><infon key="pub-id_pmid">9325247</infon><infon key="name_0">surname:Shaywitz;given-names:DA</infon><infon key="volume">272</infon><infon key="year">1997</infon><infon key="type">ref</infon><infon key="fpage">25413</infon><infon key="lpage">25416</infon><offset>72345</offset><text>COPII subunit interactions in the assembly of the vesicle coat</text></passage><passage><infon key="source">Genetics</infon><infon key="name_1">surname:Hieter;given-names:P</infon><infon key="pub-id_pmid">2659436</infon><infon key="name_0">surname:Sikorski;given-names:RS</infon><infon key="volume">122</infon><infon key="year">1989</infon><infon key="type">ref</infon><infon key="fpage">19</infon><infon key="lpage">27</infon><offset>72408</offset><text>A system of shuffle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. </text></passage><passage><infon key="source">J Biol Chem</infon><infon key="name_3">surname:Goffeau;given-names:A</infon><infon key="name_2">surname:Thines-Sempoux;given-names:D</infon><infon key="name_1">surname:Wach;given-names:A</infon><infon key="pub-id_pmid">8396147</infon><infon key="name_0">surname:Supply;given-names:P</infon><infon key="volume">268</infon><infon key="year">1994</infon><infon key="type">ref</infon><infon key="fpage">19753</infon><infon key="lpage">19759</infon><offset>72533</offset><text>Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae. </text></passage><passage><infon key="source">Cell</infon><infon key="name_3">surname:Young;given-names:R</infon><infon key="name_2">surname:Chao;given-names:DM</infon><infon key="name_1">surname:Koleske;given-names:AJ</infon><infon key="pub-id_pmid">8324825</infon><infon key="name_0">surname:Thompson;given-names:CM</infon><infon key="volume">73</infon><infon key="year">1993</infon><infon key="type">ref</infon><infon key="fpage">1361</infon><infon key="lpage">1375</infon><offset>72645</offset><text>A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast</text></passage><passage><infon key="source">EMBO (Eur Mol Biol Organ) J</infon><infon key="name_2">surname:Futcher;given-names:B</infon><infon key="name_1">surname:Tokiwa;given-names:G</infon><infon key="pub-id_pmid">8387915</infon><infon key="name_0">surname:Tyers;given-names:M</infon><infon key="volume">12</infon><infon key="year">1993</infon><infon key="type">ref</infon><infon key="fpage">1955</infon><infon key="lpage">1968</infon><offset>72744</offset><text>Comparison of the Saccharomyces cerevisiaeG1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2, and other cyclins</text></passage><passage><infon key="source">Yeast</infon><infon key="name_1">surname:Serrano;given-names:R</infon><infon key="pub-id_pmid">2528864</infon><infon key="name_0">surname:Vallejo;given-names:CG</infon><infon key="volume">5</infon><infon key="year">1989</infon><infon key="type">ref</infon><infon key="fpage">307</infon><infon key="lpage">319</infon><offset>72866</offset><text>Physiology of mutants with reduced expression of plasma membrane H+-ATPase</text></passage><passage><infon key="source">J Biol Chem</infon><infon key="name_3">surname:Ferguson;given-names:C</infon><infon key="name_4">surname:Serrano;given-names:R</infon><infon key="name_2">surname:Berberian;given-names:GE</infon><infon key="name_1">surname:Palmgren;given-names:MG</infon><infon key="pub-id_pmid">1534807</infon><infon key="name_0">surname:Villalba;given-names:JM</infon><infon key="volume">267</infon><infon key="year">1992</infon><infon key="type">ref</infon><infon key="fpage">12341</infon><infon key="lpage">12349</infon><offset>72941</offset><text>Functional expression of plant plasma membrane H(+)-ATPase in yeast endoplasmic reticulum</text></passage></document></collection>