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+<?xml version='1.0' encoding='UTF-8'?><!DOCTYPE collection SYSTEM "BioC.dtd"><collection><source>PMC</source><date>20140719</date><key>sentence.key</key><document><id>2175219</id><passage><infon key="name_3">surname:Winey;given-names:Mark</infon><infon key="name_2">surname:Giddings;given-names:Thomas H.;suffix:Jr.</infon><infon key="name_1">surname:Rout;given-names:Michael P.</infon><infon key="article-id_pmid">9864355</infon><infon key="name_0">surname:Chial;given-names:Heidi J.</infon><infon key="fpage">1789</infon><infon key="issue">7</infon><infon key="year">1998</infon><infon key="article-id_pmc">2175219</infon><infon key="type">front</infon><infon key="volume">143</infon><infon key="journal-title">The Journal of Cell Biology</infon><infon key="kwd">yeast Ndc1p spindle pole body nuclear pore complex Pom152p</infon><infon key="lpage">1800</infon><offset>0</offset><text>Saccharomyces cerevisiae Ndc1p Is a Shared Component of Nuclear Pore Complexes and Spindle Pole Bodies </text></passage><passage><infon key="type">abstract</infon><offset>104</offset><text>We report a novel connection between nuclear pore complexes (NPCs) and spindle pole bodies  (SPBs) revealed by our studies of the Saccharomyces  cerevisiae NDC1 gene. Although both NPCs and SPBs  are embedded in the nuclear envelope (NE) in yeast,  their known functions are quite distinct. Previous work  demonstrated that NDC1 function is required for  proper SPB duplication (Winey, M., M.A. Hoyt, C. Chan, L. Goetsch, D. Botstein, and B. Byers. 1993. J. Cell Biol. 122:743-751). Here, we show that Ndc1p is  a membrane protein of the NE that localizes to both  NPCs and SPBs. Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC  component, Nup49p. Additionally, distinct spots of  Ndc1p localization colocalize with a known SPB component, Spc42p. Immunoelectron microscopy shows  that Ndc1p localizes to the regions of NPCs and SPBs  that interact with the NE. The NPCs in ndc1-1 mutant  cells appear to function normally at the nonpermissive  temperature. Finally, we have found that a deletion of  POM152, which encodes an abundant but nonessential  nucleoporin, suppresses the SPB duplication defect associated with a mutation in the NDC1 gene. We show  that Ndc1p is a shared component of NPCs and SPBs  and propose a shared function in the assembly of these  organelles into the NE.</text><annotation id="0"><infon key="type">ExperimentalMethod</infon><infon key="PSIMI">0416</infon><location offset="684" length="160"/><text>Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC  component, Nup49p.</text></annotation></passage><passage><infon key="type">paragraph</infon><offset>1487</offset><text>The nuclear envelope (NE)1 remains intact throughout all stages of the cell cycle in the budding yeast,  Saccharomyces cerevisiae. Two organelles, nuclear  pore complexes (NPCs) and spindle pole bodies (SPBs),  are embedded within the NE. Beyond their common localization to the NE, these organelles have not been thought  of previously as related structures.</text></passage><passage><infon key="type">paragraph</infon><offset>1847</offset><text>NPCs are macromolecular structures that function as  the sole means by which molecules are exchanged between the nucleus and the cytoplasm. NPCs display eightfold symmetry, and are embedded within reflexed membrane pores in the NE. More than 30 NPC-associated  proteins have been identified in S. cerevisiae using a combination of biochemical and genetic approaches (reviewed  in Doye and Hurt, 1997; Wente et al., 1997). Mutations in  NPC components can affect nuclear transport, NPC distribution, NE morphology, and even spindle morphology.</text></passage><passage><infon key="type">paragraph</infon><offset>2390</offset><text>Pom152p was identified biochemically as a transmembrane glycoprotein that localizes to yeast NPCs (Wozniak  et al., 1994). Although POM152 encodes one of the most  abundant NPC components, this gene is not essential in  yeast. However, POM152 becomes essential when the null  allele is combined with mutations in a number of genes  that encode NPC components (Aitchison et al., 1995b;  Nehrbass et al., 1996). Pom152p localizes to the yeast  NPC membrane ring structure (Strambio-de-Castillia et al.,  1995; Yang et al., 1998).</text></passage><passage><infon key="type">paragraph</infon><offset>2918</offset><text>SPBs are estimated to be 20 times larger than NPCs in  diploid yeast cells (Bullitt et al., 1997). The SPB functions  as the centrosome equivalent organelle in yeast, serving as  the sole microtubule organizing center of the cell (reviewed in Snyder, 1994; Winsor and Schiebel, 1997). The  SPB is embedded in the NE where it nucleates both cytoplasmic and nuclear microtubules. Yeast cells contain a  single SPB at the beginning of G1 that must be precisely  duplicated once during each cell cycle to form the two  poles of the mitotic spindle. Several genes have been identified in which mutations affect specific steps in SPB duplication (reviewed in Winey and Byers, 1993).</text></passage><passage><infon key="type">paragraph</infon><offset>3595</offset><text>The S. cerevisiae NDC1 gene has been shown to function at a late step in SPB duplication (Winey et al., 1993).  Although SPB duplication is initiated in ndc1-1 strains at  the nonpermissive temperature, the newly synthesized  SPB is not inserted into the NE. The defective SPB remains on the cytoplasmic face of the NE where it nucleates cytoplasmic microtubules, but it fails to nucleate nuclear microtubules. The preexisting SPB is functional in  these cells, and all of the chromosomes remain associated  with it. Thus, ndc1-1 cells arrest with large buds and a G2  DNA content in response to their monopolar spindles.  Eventually, these cells undergo asymmetric cell division in  which all of their chromosomal DNA segregates with the  single, functional SPB; as a result, one cell doubles in  ploidy and the other cell lacks chromosomal DNA (Thomas and Botstein, 1986). NDC1 encodes an essential 74-kD protein with six to seven potential transmembrane domains (Winey et al., 1993). Previous attempts to examine  Ndc1p localization using overexpressed protein revealed  perinuclear staining (Winey et al., 1993).</text></passage><passage><infon key="type">paragraph</infon><offset>4712</offset><text>We show here that Ndc1p expressed at endogenous levels localizes to both NPCs and SPBs. Additionally, we  have found that a deletion of POM152 suppresses the  ndc1-1 SPB duplication defect. Our results uncover a previously unknown link between two NE-embedded organelles, NPCs and SPBs.</text></passage><passage><infon key="type">title_1</infon><offset>4999</offset><text>Materials and Methods</text></passage><passage><infon key="type">title_2</infon><offset>5021</offset><text>Yeast Strains and Media</text></passage><passage><infon key="type">paragraph</infon><offset>5045</offset><text>Relevant yeast strains used in this study are listed in Table I and were constructed using standard techniques (Sherman et al., 1986). The chromosomal copy of NDC1 was epitope tagged at its 3' end by creating an in-frame fusion to the IgG binding domains of protein A (ProA) as described  by Aitchison et al. (1995a). NDC1-ProA strains grew at all temperatures  ranging from 15 to 37 C and exhibited stable ploidy. The ndc1-1, pom152  null strain [HC5-31c(1166); Table I] was constructed by replacing the entire POM152 open reading frame, plus an additional 163 bp after the stop  codon, with the HIS3 gene (Baudin et al., 1993). NDC1-ProA strains containing NUP49-GFP (HC26-15a/1a; Table I) were constructed by crossing a NDC1-ProA strain to yeast strain SWY809 (Bucci and Wente, 1997)  that contains nup49-1::URA3 and nup49DeltaGFLG::GFP-S65T-TRP1.  Strains containing NDC1-ProA, NUP49-GFP, and the nup133 null allele  (HC27-16d/16b; Table I) were constructed by crossing a NDC1-ProA  strain to yeast strain SWY828 (Bucci and Wente, 1998) that contains  nup133Delta::HIS3, nup49-1::URA3, and nup49DeltaGFLG::GFP-S65T-TRP1. A  yeast strain containing NIC96-ProA was constructed as described by  Aitchison et al. (1995a). Yeast strains containing either NDC1-ProA or  NIC96-ProA with SPC42-GFP (HC22-2b/1c and HC23-11d/16a, respectively) were constructed by crossing to yeast strain IAY18 (a kind gift  from Ian Adams and John Kilmartin) that contains spc42Delta::LEU2 and  TRP1::SPC42-GFP(3x) (Schutz and Winey, 1998). Yeast strain HC14-10c(1198)/HC29-6b is homozygous for two different null alleles of NDC1;  the entire NDC1 open reading frame was removed in both cases. The  ndc1Delta::HIS3 null allele was made by integrative transformation of a PCR  product containing the HIS3 gene (Baudin et al., 1993). The ndc1Delta:: KANMX null allele was constructed using a two-step gene disruption  technique (Rothstein, 1991); in this case, the NDC1 open reading frame  was replaced with the KANMX gene (Wach et al., 1994). </text></passage><passage><infon key="type">paragraph</infon><offset>7052</offset><text>All yeast strains were grown as described, in YPD (1% yeast extract,  2% bactopeptone, 2% glucose), YPR (1% yeast extract, 2% bactopeptone, 3% raffinose), synthetic media supplemented with the appropriate  amino acids and 2% glucose, or synthetic media supplemented with the  appropriate amino acids and 3% raffinose. We used YPR containing 2%  galactose for galactose induction. Yeast strains were grown at the temperatures indicated. Techniques for yeast manipulation were carried out as  described by Sherman et al. (1986). Cells were arrested in G1 using alpha-factor obtained from a custom peptide synthesis that was HPLC purified  (Macromolecular Resources, Ft. Collins, CO), at a concentration of 11 mug/ml  for 2 h.</text></passage><passage><infon key="type">title_2</infon><offset>7776</offset><text>Plasmids</text></passage><passage><infon key="type">paragraph</infon><offset>7785</offset><text>Plasmids used in this study were constructed and introduced into Escherichia coli or yeast using standard techniques (Ausubel et al., 1994).  pALR10-NDC1 contains an AgeI-SacI NDC1 fragment cloned into the  XmaI/SalI and SacI sites of the pALR10 vector (a kind gift from Alain  Camasses), a modified version of the centromeric plasmid pUN50  (Elledge and Davis, 1988) that contains the URA3 and ADE3 selectable  markers. The pGAL-NIC96-ProA plasmid contains NIC96-ProA that by  PCR amplification using genomic DNA from yeast strain HC13-1c (Table  I) contained BglII sites on each end cloned into the BamHI site of the  pBM272 vector, just after the GAL1 promoter (Johnston and Davis,  1984). The pRS315-NDC1-green fluorescent protein (GFP) plasmid was  constructed by subcloning a PCR fragment containing GFP amplified  from the pyEGFP3 plasmid (Cormack et al., 1997) with XbaI restriction  sites into an AvrII linker introduced at the end of NDC1 cloned into the  pRS315 vector (Sikorski and Hieter, 1989).</text></passage><passage><infon key="type">title_2</infon><offset>8794</offset><text>Protein Techniques</text></passage><passage><infon key="type">paragraph</infon><offset>8813</offset><text>Fractionation of yeast strains was carried out as described previously  (Rout and Kilmartin, 1990; Rout and Kilmartin, 1991; Rout and Blobel,  1993; Strambio-de-Castillia et al., 1995; Rout and Strambio-de-Castillia,  1998) using yeast strain ProA5-4d (Table I). Extractions of the isolated  NEs were carried out using 0.1 M sodium carbonate, pH 11.5, as described  previously (Wozniak et al., 1994). Peptide sequencing was carried out as  described previously (Fernandez et al., 1994). Ndc1p-ProA and Pom152p  were detected using purified rabbit IgG (1:1,000; ICN Pharmaceuticals,  Inc., Costa Mesa, CA) and mAb 118C3 (1:5, culture supernatant) (Strambio-de-Castillia et al., 1995), respectively. Spc110p was detected using a  combination of three monoclonal supernatants: 3D2, 36G6, and 45D10  (1:20, culture supernatants) (Rout and Kilmartin, 1990). Nup57p was detected using a mixture of mAb 414 and mAb 350 (1:5, culture supernatants) (Davis and Blobel, 1986; Davis and Blobel, 1987; Davis and Fink,  1990).</text></passage><passage><infon key="type">title_2</infon><offset>9826</offset><text>Cytological Techniques</text></passage><passage><infon key="type">paragraph</infon><offset>9849</offset><text>Buffer solutions used for immunofluorescence (IF) microscopy include  PBSA (10 mg/ml NaCl, 0.2 mg/ml KCl, 1.43 mg/ml KH2PO4), solution A  (1.2 M sorbitol, 100 mM KPO4, pH 7.5), spheroplasting solution (solution  A containing 4.83 mug/ml Zymolyase 100T, 0.1 M beta-mercaptoethanol), and  blocker (PBSA containing 10 mg/ml BSA, 0.1% Tween 20).</text><annotation id="1"><infon key="type">ExperimentalMethod</infon><infon key="PSIMI">0416</infon><location offset="9849" length="341"/><text>Buffer solutions used for immunofluorescence (IF) microscopy include  PBSA (10 mg/ml NaCl, 0.2 mg/ml KCl, 1.43 mg/ml KH2PO4), solution A  (1.2 M sorbitol, 100 mM KPO4, pH 7.5), spheroplasting solution (solution  A containing 4.83 mug/ml Zymolyase 100T, 0.1 M beta-mercaptoethanol), and  blocker (PBSA containing 10 mg/ml BSA, 0.1% Tween 20).</text></annotation></passage><passage><infon key="type">paragraph</infon><offset>10189</offset><text>IF microscopy of proteins epitope-tagged with the IgG binding domains of ProA was carried out as follows. Log phase growing cells were  fixed for 5 min using formaldehyde (3.7% in PBSA), followed by rinsing  with PBSA. The cells were then resuspended in spheroplasting solution  and incubated at 30 C for 45 min, followed by rinsing with PBSA and resuspension in solution A. Spheroplasted cells were adhered to poly-lysine-treated slides. The slides were submerged into chilled 100% methanol on ice, allowed to equilibrate to room temperature, and immersed into  100% acetone at room temperature for 30 s. Dried slides were treated  with blocker, and primary antibody (rabbit IgG, 2 mg/ml in 0.15 M NaCl  diluted 1:2,000; Sigma Chemical Co., St. Louis, MO) was applied overnight. After rinses with PBSA, the secondary antibody was applied; either  FITC-conjugated donkey anti-rabbit IgGs (1:100; Amersham Life Science  Inc., Arlington Heights, IL) or Texas red-conjugated goat anti-rabbit  IgGs (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove,  PA) were used. The slides were rinsed with PBSA and were treated with  4',6-diamidino-2-phenylindole (Sigma Chemical Co.) to stain DNA. The  slides were mounted in Citifluor (Ted Pella, Inc., Redding, CA).</text></passage><passage><infon key="type">paragraph</infon><offset>11465</offset><text>Standard fluorescence microscopy was carried out using a Zeiss fluorescence microscope equipped with an Empix CCD camera. Images were  captured using MetaMorph imaging software (Universal Imaging, West  Chester, PA). Deconvolution microscopy was carried out using a Leica  DMRXA/RF4/V automated universal microscope equipped with a Cooke  SensiCam high performance digital camera. Images were acquired and deconvolved using the Slidebook software package (Intelligent Imaging Innovations, Denver, CO).</text><annotation id="2"><infon key="type">ExperimentalMethod</infon><infon key="PSIMI">0416</infon><location offset="11465" length="121"/><text>Standard fluorescence microscopy was carried out using a Zeiss fluorescence microscope equipped with an Empix CCD camera.</text></annotation></passage><passage><infon key="type">paragraph</infon><offset>11967</offset><text>Immunoelectron microscopy was carried out as described previously  (Ding et al., 1997). Approximately 5-ml aliquots of yeast cultures were  harvested by vacuum filtration using 0.45-mum Milipore filters, and were  high pressure frozen in a Balzers HPM-010 High Pressure Freezer  (Winey et al., 1995). Samples were freeze substituted in 0.15% glutaraldehyde in acetone at -80 C for 3 d, gradually warmed to 4 C, and embedded in LR White resin. Thin sections were labeled with an anti-GFP  rabbit polyclonal antiserum (a kind gift from Jason Kahana and Pamela  Silver, Dana-Farber Cancer Institute). Sections were mounted on formvar-coated nickel grids and were incubated in 0.1 N HCl for 10 min, 50  mM ammonium acetate for 15 min, and in blocking buffer (0.8% BSA,  0.1% gelatin, 50 mM sodium phosphate, pH 7.2, 150 mM NaCl, and 0.1%  Tween 20) for 1 h. The grids were then floated for 2 h on drops of primary  antibody diluted 1:100 in blocking buffer, followed by rinses with PBST  (50 mM sodium phosphate, pH 7.2, 150 mM NaCl, and 0.1% Tween 20).  The grids were then reacted with goat anti-rabbit 15-nm colloidal gold  conjugate (Ted Pella, Inc.) diluted 1:20 in blocking buffer for 1 h, followed  by rinses and staining with uranyl acetate and lead citrate.</text></passage><passage><infon key="type">paragraph</infon><offset>13236</offset><text>Flow cytometry was carried out as described (Hutter and Eipel, 1979)  using propidium iodide to stain DNA (Sigma Chemical Co.). Stained cells  were analyzed using a FACScan  flow cytometer and the Cell Quest software package for data analysis (Becton Dickinson, San Jose, CA).</text></passage><passage><infon key="type">title_2</infon><offset>13514</offset><text>Assays to Examine NPC Assembly</text></passage><passage><infon key="type">paragraph</infon><offset>13545</offset><text>Assays to examine NPC assembly were carried out using yeast strains  HC10-42b/42c and HC10-42a/42d (Table I) transformed with pGAL-NIC96-ProA. Asynchronous cultures grown in 30 C Ura- raffinose media  were spun down and resuspended in prechilled YPR media. After 18 h,  prechilled galactose was added to the cultures to a 2% final concentration.  NIC96-ProA expression was induced for 12 h, followed by a glucose chase  for 4 h. Throughout these experiments, samples were taken for IF microscopy, flow cytometry, and budding index analysis. In a separate assay,  NPC numbers were determined directly using electron micrographs of serially sectioned cells with computer modeling techniques described by  Winey et al. (1997) using yeast strains Wx340-3c and Wx340-3d (Table I).</text></passage><passage><infon key="type">title_1</infon><offset>14324</offset><text>Results</text></passage><passage><infon key="type">title_2</infon><offset>14332</offset><text>Ndc1p Localizes to NPCs</text></passage><passage><infon key="type">paragraph</infon><offset>14356</offset><text>We used a previously published biochemical fractionation  technique to isolate highly enriched NPCs from Saccharomyces (Rout and Blobel, 1993). Individual protein components of the enriched NPCs were identified by direct  peptide sequence analysis. One of the peptide sequences  obtained corresponded exactly to the first 21 NH2-terminal amino acids of Ndc1p (MIQTPRELLNPRYTYHTIFSD) (Winey et al., 1993). Additionally, the protein  band from which this peptide sequence was obtained corresponded in molecular weight to Ndc1p and was recognized by anti-Ndc1p antibodies (data not shown).</text></passage><passage><infon key="type">paragraph</infon><offset>14943</offset><text>We tested whether Ndc1p from S. cerevisiae localizes to  NPCs using a strain that contains NDC1 epitope-tagged  at its COOH terminus with the IgG binding domains of  ProA (NDC1-ProA). This epitope-tagged version of NDC1  is chromosomally integrated replacing the original copy of  NDC1, is expressed using the endogenous NDC1 promoter, and is fully functional (data not shown). We used  indirect IF deconvolution microscopy to examine the localization of Ndc1p-ProA in whole cells and found that it  displayed punctate nuclear peripheral staining, suggestive  of NPC localization (Fig. 1 A) (Rout and Wente, 1994).  We saw similar localization using strains with a chromosomally integrated version of NDC1 that contains three  repeats of the c-myc epitope at its COOH terminus (data  not shown). </text></passage><passage><infon key="type">paragraph</infon><offset>15740</offset><text>We then tested whether Ndc1p-ProA colocalizes with a  known NPC component, Nup49p (Wente et al., 1992). We  used strains that contain both NDC1-ProA and an allele of  NUP49 that is epitope-tagged with the Aequorea victoria  GFP (Bucci and Wente, 1997). When Ndc1p-ProA was indirectly labeled with Texas red, we observed colocalization with Nup49p-GFP signal (Fig. 1 B). It is important  that this colocalization relied on the autofluorescence of  Nup49p-GFP, because the ProA epitope on Ndc1p can  cross-react with any other antibody used for double immunolabeling. We also examined this colocalization in a  nup133 null strain in which NPCs become clustered (Doye  et al., 1994; Pemberton et al., 1995). Ndc1p-ProA localization became clustered and colocalized with clusters of  Nup49p-GFP signal in this strain (Fig. 1 C), suggesting  that Ndc1p-ProA localizes to NPCs and not merely to the  NE. Occasionally, we observed either one or two additional bright spots of Ndc1p-ProA staining that did not  colocalize with Nup49p-GFP signal (see below).</text></passage><passage><infon key="type">paragraph</infon><offset>16790</offset><text>We also carried out biochemical fractionation experiments to isolate NPCs and NEs from strains that contain  Ndc1p-ProA. In both cases, Ndc1p-ProA cofractionated  with the known NPC marker, Pom152p (Fig. 2, A and B).  We also examined these fractions using monoclonal antibodies recognizing numerous repeat motif-containing  nucleoporins (Wente et al., 1997) and observed similar  fractionation patterns (data not shown). Hydrophobicity  plots of Ndc1p predict that it contains six to seven transmembrane domains (Winey et al., 1993). We extracted the  NE fraction with carbonate to test whether Ndc1p-ProA  behaves as an integral membrane protein. After centrifugation, soluble NPC components, such as Nup57p, were  extracted by carbonate (Fig. 2 C). In contrast, Pom152p, a  known integral membrane protein of the NE, remained in  the pellet (Fig. 2 C). Ndc1p-ProA was not extracted from  the NE by carbonate treatment (Fig. 2 C), suggesting that  it is an integral membrane protein of the NE. Therefore,  in combination with the whole cell IF localization data,  these results indicate that Ndc1p-ProA is an integral membrane protein of NPCs. </text></passage><passage><infon key="type">title_2</infon><offset>17937</offset><text>Ndc1p Also Localizes to SPBs</text></passage><passage><infon key="type">paragraph</infon><offset>17966</offset><text>Because NDC1 function is required for proper SPB duplication, we tested whether Ndc1p also localizes to SPBs.  This analysis was difficult because both NPCs and SPBs  localize to the NE, and cells contain either one or two  SPBs in the midst of 100-150 NPCs. We addressed this  question using several experimental approaches. We used  a biochemical fractionation technique to isolate SPBs from  cells containing Ndc1p-ProA (Rout and Kilmartin, 1990).  Immunoblotting of isolated fractions showed that Ndc1p-ProA partially cofractionated with SPBs (Fig. 2 A). However, one concern about these fractionation experiments  was the heavy contamination of NPCs in the SPB fraction,  making it difficult to distinguish the fractionation pattern  of a protein that was only in the NPC from that observed  for Ndc1p-ProA.</text></passage><passage><infon key="type">paragraph</infon><offset>18781</offset><text>Therefore, we used whole cell indirect IF deconvolution  microscopy to test whether any of the distinct spots of  Ndc1p staining colocalize with signal from a known SPB  component. This high-resolution fluorescence microscopy  technique could potentially resolve SPB- and NPC-associated signals in the yeast NE. We used a strain that contains  both NDC1-ProA and SPC42-GFP in these experiments.  SPC42 encodes an essential SPB component that forms a  crystalline layer between the central and outer plaque of  the SPB (Rout and Kilmartin, 1991; Donaldson and Kilmartin, 1996; Bullitt et al., 1997). Additionally, we used a  strain that contains both NIC96-ProA and SPC42-GFP as  a negative control to ensure that overlapping signal was  not merely due to a lack of resolution. Nic96p is known to  localize to NPCs (Grandi et al., 1993), but it has been characterized extensively and is unlikely to localize to SPBs as  well (Grandi et al., 1993; Aitchison et al., 1995b; Grandi  et al., 1995; Zabel et al., 1996; Schlaich et al., 1997; Ho et al.,  1998).</text><annotation id="3"><infon key="type">ExperimentalMethod</infon><infon key="PSIMI">0416</infon><location offset="18781" length="311"/><text>Therefore, we used whole cell indirect IF deconvolution  microscopy to test whether any of the distinct spots of  Ndc1p staining colocalize with signal from a known SPB  component. This high-resolution fluorescence microscopy  technique could potentially resolve SPB- and NPC-associated signals in the yeast NE.</text></annotation></passage><passage><infon key="type">paragraph</infon><offset>19836</offset><text>We were able to distinguish between NPC and SPB signals using this technology. We observed a distinct spot of  Ndc1p-ProA staining that colocalized with Spc42p-GFP  signal in 91% of the SPBs examined (n = 63) (Fig. 3 A). In  contrast, Nic96p-ProA staining did not exhibit colocalization with Spc42p-GFP signal in 82% of the SPBs examined  (n = 51) (Fig. 3 B). Both Ndc1p and Nic96p contained the  ProA epitope tag, allowing us to use the same antibodies  to detect their localization. Additionally, the localization of  Spc42p-GFP was visualized using GFP autofluorescence  and therefore did not rely on potentially cross-reacting antibodies. </text></passage><passage><infon key="type">paragraph</infon><offset>20480</offset><text>As described earlier, when examining the colocalization  of Ndc1p-ProA with Nup49p-GFP in a NPC clustering  mutant strain, we occasionally observed either one or two  distinct spots of Ndc1p-ProA staining that did not overlap  with Nup49p-GFP signal (Fig. 3 C). It is likely that these  additional spots of Ndc1p-ProA staining correspond to  SPB localization. Previous work demonstrated that the  SPB often localizes within NPC clusters (Heath et al.,  1995), explaining why we observed extra spots of Ndc1p-ProA staining only occasionally.</text></passage><passage><infon key="type">paragraph</infon><offset>21021</offset><text>Finally, we examined the localization of Ndc1p-GFP in  living cells. Although these cells displayed punctate nuclear peripheral localization of Ndc1p-GFP signal, they  also contained either one or two intense spots of Ndc1p-GFP signal (Fig. 4 A). We propose that these bright spots  correspond to SPB localization based on three criteria.  First, SPBs are estimated to be 20 times larger than NPCs  in diploid yeast cells (Bullitt et al., 1997), explaining why  the signal might appear larger and brighter. Second, we  observed either one or two bright spots of Ndc1p-GFP signal, corresponding to the number of SPBs normally found  in cells. Finally, the bright spots were present at positions  in the cell that are consistent with SPB localization. For  example, the late mitotic cells displayed a bright spot at  each end of the NE, where the SPBs are normally located.  These bright spots were present in all of the cells we examined, suggesting that Ndc1p-GFP is associated with the  SPB at all stages of the cell cycle. </text></passage><passage><infon key="type">title_2</infon><offset>22047</offset><text>Immunoelectron Microscopy Demonstrates that Ndc1p-GFP Localizes to the Membrane-associated  Regions of NPCs and SPBs</text></passage><passage><infon key="type">paragraph</infon><offset>22164</offset><text>Although the experiments using whole cell IF microscopy  provide strong evidence that Ndc1p localizes to both  NPCs and SPBs, we also used immunoelectron microscopy  to more closely examine the localization of Ndc1p-GFP  within these two organelles. These experiments were carried out using a yeast strain that contains NDC1-GFP.  Consistent with previous data, we observed immunogold  labeling, corresponding to the localization of Ndc1p-GFP,  at both NPCs and SPBs using this technique. Immunogold  labeling was most often seen at the periphery of the NPCs  (Fig. 4 B) and at the edges of the central plaque of the SPB  (Fig. 4 C); these are the regions of NPCs and SPBs that  interact with the NE. A similar localization was found  for Ndc1p-ProA in biochemically isolated SPBs from the  Ndc1p-ProA-containing strain using immunoelectron microscopy (data not shown).</text></passage><passage><infon key="type">title_2</infon><offset>23036</offset><text>ndc1-1 Strains Do Not Exhibit Defects in NPC Functions</text></passage><passage><infon key="type">paragraph</infon><offset>23091</offset><text>A role for Ndc1p at SPBs has been established clearly  (Winey et al., 1993); however, the localization of Ndc1p to  NPCs was quite unexpected. We carried out assays to determine whether Ndc1p plays a role in NPC function. We  tested whether ndc1-1 cells exhibit defects in NPC-related  functions at the nonpermissive temperature. ndc1-1 strains  arrest as large budded cells with a G2 DNA content after a  shift to the nonpermissive temperature (Thomas and Botstein, 1986; Winey et al., 1993). This arrest indicates that  NDC1 function, at least with respect to SPB duplication,  has been lost (Winey et al., 1993). We examined NPC phenotypes at this arrest to ensure that ndc1-1 function was  minimal.</text></passage><passage><infon key="type">paragraph</infon><offset>23794</offset><text>We did not detect any defects in nuclear protein import  in ndc1-1 strains after inducing the expression of histone  H2B1 as a nuclear localization signal (NLS) fused to GFP  (pJON280; Schlenstedt et al., 1995) at the ndc1-1 arrest  (data not shown). Because the H2B1p-GFP fusion protein  was detectable in ndc1-1 cells at the arrest, it seemed likely  that its corresponding mRNA was properly synthesized  and exported from the nucleus. We carried out a similar  experiment using the same yeast strains transformed with  a different plasmid containing a NLS derived from the  yeast ribosomal protein L29 fused to beta-galactosidase as a  reporter expressed under the control of the GAL10 promoter  (pNLS-E1; Underwood and Fried, 1990). Induced protein  levels were detected by immunoblotting, and beta-galactosidase expression was similar in both ndc1-1 and wild-type  cells (data not shown), indicating that mRNA export was  likely to be occurring properly at the ndc1-1 arrest. Finally, we examined NPC clustering, a phenotype observed  when specific nucleoporins are mutated or deleted (Doye  et al., 1994; Aitchison et al., 1995a; Heath et al., 1995; Pemberton et al., 1995). We did not observe any clustering of  NPCs in ndc1-1 strains using Nic96p localization as a marker  for NPCs (Grandi et al., 1993) (data not shown).</text></passage><passage><infon key="type">paragraph</infon><offset>25120</offset><text>Because ndc1-1 cells are defective in the assembly of  their new SPB into the NE, we examined whether ndc1-1  cells also exhibit defects in the assembly of newly synthesized NPCs into the NE. We tested whether ndc1-1 strains  properly assemble an epitope-tagged version of Nic96p  into their NE at the ndc1-1 arrest. Wild-type or ndc1-1  strains were transformed with a plasmid that contains  NIC96-ProA expressed under the control of a galactose-inducible promoter. Thus, any ProA signal observed was  due to induced Nic96p-ProA expression. These cells did  contain their endogenous untagged chromosomal copy of  NIC96, because this gene is essential (Grandi et al., 1993).  Transformed yeast strains were shifted to the nonpermissive temperature of 15 C, and NIC96-ProA expression was  induced at the ndc1-1 arrest (Fig. 5, 18h). The fusion protein was present throughout the cell as seen by indirect IF  microscopy after 12 h of induction (Fig. 5, 30h). We then  added glucose to the cultures to turn off further expression  of Nic96p-ProA, allowing us to test whether the induced  protein was assembled into NPCs. Nic96p-ProA was properly assembled into the NE in both wild-type and ndc1-1  cells (Fig. 5, 34h). </text></passage><passage><infon key="type">paragraph</infon><offset>26338</offset><text>We further examined NPC assembly functions by using  a recently published technique to directly count the number of NPCs in ndc1-1 cells at the nonpermissive temperature (Winey et al., 1997). We determined the number of  NPCs in individual nuclei by generating computer-aided  reconstructions of whole nuclei from electron micrographs  of serially sectioned cells. The defective SPB in ndc1-1  cells is still capable of nucleating cytoplasmic MTs, allowing it to migrate and carry with it a thin finger of NE  (Winey et al., 1993). This deformed NE is difficult to follow through serial sections. The tub2-403 allele is a cold-sensitive mutation in beta-tubulin (Huffaker et al., 1988) that  prevents the defective ndc1-1 SPB from migrating (Winey,  M., unpublished observation). We used a ndc1-1, tub2-403  double mutant strain (Wx340-3c; Table I) for this analysis,  and a strain containing the tub2-403 allele alone (Wx340-3d; Table I) as a control for mitotically arrested cells (Huffaker et al., 1988).</text></passage><passage><infon key="type">paragraph</infon><offset>27344</offset><text>Samples were prepared as previously described (Winey  et al., 1997) to directly count the number of NPCs in three  nuclei from each strain after a shift to the nonpermissive  temperature. Nuclei from the tub2-403 cells contained 162,  179, and 232 NPCs, and nuclei from the ndc1-1, tub2-403  double mutant cells contained 180, 193, and 209 NPCs.  The maximum number of NPCs observed in wild-type  cells occurs in early mitosis, with an average of 142 +- 16.4  NPCs (Winey et al., 1997). Our analysis demonstrated that  both strains contained more NPCs than observed in wild-type cells at any point in the cell cycle, indicating that NPC  assembly continues to occur when these cells are arrested.</text></passage><passage><infon key="type">title_2</infon><offset>28041</offset><text>A Deletion of POM152 Suppresses the ndc1-1 SPB  Duplication Defect</text></passage><passage><infon key="type">paragraph</infon><offset>28108</offset><text>To define a potential functional role for Ndc1p in NPCs,  we examined genetic interactions between NDC1 and  POM152. We were particularly interested in POM152 because it encodes an integral membrane protein that localizes to the pore membrane domain of NPCs (Wozniak et  al., 1994). Although POM152 encodes an abundant nucleoporin, this gene is not essential. We initially examined  potential synthetic lethal interactions between ndc1-1 and  the pom152 null allele, but no synthetic lethality was observed when these two mutations were combined.</text></passage><passage><infon key="type">paragraph</infon><offset>28655</offset><text>Unexpectedly, we found that the pom152 null allele suppressed the ndc1-1 cold-sensitive phenotype. ndc1-1 strains  containing the pom152 null allele were able to grow on  plates incubated at 15 C, the nonpermissive temperature  for ndc1-1 (Fig. 6 A). This suppression was due specifically  to a loss of POM152, because when a plasmid-borne copy  of POM152 was reintroduced into the double mutant  strain, the ndc1-1 cold-sensitive phenotype was restored  (Fig. 6 A). Additionally, this suppression is specific for the  ndc1-1 allele, because the pom152 null allele could not  function as a bypass suppressor of the ndc1 null allele  (data not shown). </text></passage><passage><infon key="type">paragraph</infon><offset>29308</offset><text>NDC1 function is required for SPB duplication in the  first cell cycle after release from a G1 arrest induced  by treatment with alpha-factor (Winey et al., 1993). We  tested whether the deletion of POM152 suppresses the  ndc1-1 SPB duplication defect in the first cell cycle after a  shift to the nonpermissive temperature. Cells were arrested  in G1 at the permissive temperature using alpha-factor,  and released into prechilled media at the nonpermissive  temperature for ndc1-1. Wild-type cells synchronously released from the G1 arrest, completed the cell cycle, and  eventually became asynchronous (Fig. 6 B). In contrast,  ndc1-1 cells synchronously released from the G1 arrest,  but then arrested with a G2 DNA content and large buds  due to failed SPB duplication in the first cell cycle (Fig. 6  B). The ndc1-1, pom152 null double mutant strains did not  exhibit the ndc1-1 cell cycle arrest; they appeared similar  to the wild-type cultures (Fig. 6 B). We conclude that the  pom152 null allele suppresses the ndc1-1 SPB duplication  defect in the first cell cycle.</text></passage><passage><infon key="type">title_1</infon><offset>30385</offset><text>Discussion</text></passage><passage><infon key="type">paragraph</infon><offset>30396</offset><text>Two organelles, SPBs and NPCs, interrupt the NE and allow communication and coordination to occur between  the nucleus and the cytoplasm. Although these organelles  differ greatly in their characterized functions, our findings  suggest that they may be more similar than previously  thought; we show here that the integral membrane protein  Ndc1p localizes to both SPBs and NPCs, the first such  shared component identified. Endogenous Ndc1p was identified biochemically as a constituent of highly enriched yeast  NPCs, and IF microscopy demonstrated that Ndc1p colocalized with a known nucleoporin.</text></passage><passage><infon key="type">paragraph</infon><offset>30996</offset><text>We were interested in determining whether Ndc1p also  localizes to SPBs because ndc1-1 cells exhibit defects in  SPB duplication (Winey et al., 1993). SPBs are greatly outnumbered by NPCs in the NE, making it difficult to assess  whether signal observed is specific to SPBs. We were able  to distinguish between NPC and SPB signal using indirect  IF deconvolution microscopy of whole cells. We identified  distinct spots of Ndc1p signal that colocalized with a SPB  marker. Control experiments using an abundant NPC component showed little colocalization with the SPB marker,  indicating that NPCs and SPBs could be resolved. This  suggests that Ndc1p is associated with the SPB, consistent  with the SPB duplication defect observed in ndc1-1 mutant strains.</text></passage><passage><infon key="type">paragraph</infon><offset>31755</offset><text>However, SPBs and NPCs are most clearly distinguished  by using electron microscopy. Examination of Ndc1p- GFP localization using immunoelectron microscopy showed  that immunogold labeling, corresponding to Ndc1p-GFP,  was concentrated at both NPCs and SPBs. The distribution of immunogold particles at the periphery of NPCs and  at the edges of the central plaque region of the SPB  showed that Ndc1p-GFP often localized to the regions of  the NPC and SPB that interact with the NE. Ultrastructural analysis of the yeast NPC shows that this organelle  contains a unique membrane interacting ring (Strambio-de-Castillia et al., 1995; Yang et al., 1998). It is possible  that the SPB contains a similar membrane interacting domain. Our results suggest that Ndc1p is a shared component of the membrane interacting regions of NPCs and  SPBs.</text><annotation id="4"><infon key="type">ExperimentalMethod</infon><infon key="PSIMI">0040</infon><location offset="31755" length="84"/><text>However, SPBs and NPCs are most clearly distinguished  by using electron microscopy.</text></annotation></passage><passage><infon key="type">paragraph</infon><offset>32594</offset><text>Interestingly, the cut11+ gene of Schizosaccharomyces  pombe, like NDC1, encodes a protein with seven potential  membrane spanning regions, and mutations in cut11+ affect SPB function (West et al., 1998). Cut11p also localizes  to NPCs and SPBs, and cut11+ and NDC1 also show limited functional homology (West et al., 1998). However, a  function for Cut11p at NPCs has not been determined.</text></passage><passage><infon key="type">paragraph</infon><offset>32984</offset><text>A function for Ndc1p at the SPB has been established  clearly from previous work; ndc1-1 cells fail to insert their  newly synthesized SPB into the NE at the nonpermissive  temperature (Winey et al., 1993). We do not observe any  defects in nuclear transport, in NPC distribution, or in  NPC assembly at the nonpermissive temperature using the  existing ndc1-1 allele (Thomas and Botstein, 1986). It is  possible that new mutant alleles of NDC1 will uncover  NPC related phenotypes. Studies of NDC1 gene dosage  show that the ndc1-1 allele does not exhibit a complete  loss of function at the nonpermissive temperature (Chial,  H.J., unpublished observation). Several NPC components  are not essential, suggesting that many nucleoporins carry  out redundant functions. It is possible that other NPC  components will have to be disrupted before a NPC-related function for NDC1 will be uncovered. NDC1 may  perform a similar role for NPCs as has been established  for SPBs, possibly being involved in their assembly into  the NE.</text></passage><passage><infon key="type">paragraph</infon><offset>34012</offset><text>Although little is known about how yeast NPCs are assembled into the NE, studies of the NIC96 gene of S. cerevisiae have shown that it is involved in the formation of  NPCs. Cells containing a conditional mutation in NIC96  exhibited decreased numbers of NPCs after exposure to  the nonpermissive temperature (Zabel et al., 1996). Recently, a vertebrate homologue of NIC96, called NUP93,  was identified (Grandi et al., 1997). After immunodepletion of Nup93p in a Xenopus nuclear reconstitution assay,  the nuclei exhibited defects in NPC assembly (Grandi et al.,  1997). Yeast Nic96p has been shown both in vivo and in  vitro to be associated with a NPC subcomplex that contains Nsp1p, Nup49p, and Nup57p, which can be assembled into NPCs (Grandi et al., 1993; Grandi et al., 1995;  Schlaich et al., 1997). Additionally, synthetic lethal interactions have been identified between mutant alleles of  NIC96, NUP188, and POM152 (Aitchison et al., 1995b;  Nehrbass et al., 1996; Zabel et al., 1996).</text></passage><passage><infon key="type">paragraph</infon><offset>35009</offset><text>Until now, only synthetic lethal interactions have been  observed when the pom152 null allele is combined with  mutations in a number of NPC components (Aitchison et al.,  1995b; Nehrbass et al., 1996). We report that the removal  of Pom152p, one of the most abundant NPC components,  strongly suppresses the ndc1-1 SPB duplication defect. In  fact, this deletion suppresses the ndc1-1 SPB duplication  defect within the first cell cycle after a shift to the nonpermissive temperature. Because a function for Ndc1p at  NPCs has not been determined, we do not know if the suppression of ndc1-1 by the pom152 null allele has any functional consequence for NPCs. Nonetheless, this suppression lends support to the idea that SPBs and NPCs are  somehow linked, perhaps in some mechanistic way beyond  shared components.</text></passage><passage><infon key="type">paragraph</infon><offset>35824</offset><text>The mechanism by which the pom152 null allele suppresses the ndc1-1 SPB duplication defect is not known,  but it appears as if wild-type POM152 is antagonistic in  ndc1-1 strains. To further understand this interaction, it  will be necessary to learn whether Pom152p, in addition to  its localization to NPCs, also localizes to SPBs. Although  the POM152 gene is not essential, the overexpression of  POM152 is toxic to yeast cells (Wozniak et al., 1994). We  tested whether the toxicity associated with POM152 overexpression is due to a defect in SPB duplication, but we  did not observe any SPB-related phenotypes in cells overexpressing POM152 (Chial, H.J., unpublished observation).</text></passage><passage><infon key="type">paragraph</infon><offset>36511</offset><text>If Pom152p localizes only to NPCs, it is possible that  a deletion of this nonessential NPC component releases  Ndc1-1p from NPCs, allowing it to function at the SPB.  This idea is supported by our observation that providing  haploid ndc1-1 cells with an additional chromosomally integrated copy of the ndc1-1 allele relieves the ndc1-1 cold-sensitive phenotype, suggesting that Ndc1-1p can function  in SPB duplication if present at slightly elevated levels in  the cell (Chial, H.J., unpublished observation). It is possible that Ndc1p and Pom152p may directly interact at  NPCs, because immunoelectron microscopy shows that  these two proteins localize to similar regions of the NPC  (Wozniak et al., 1994), and both are membrane proteins.  However, if Pom152p localizes to both NPCs and SPBs, it  is possible that Pom152p at the SPB somehow interferes  with the ability of mutant Ndc1-1p to function in the  proper insertion of the newly synthesized SPB into the  NE. In this case, the removal of Pom152p might allow  Ndc1-1p to function more efficiently at the SPB.</text></passage><passage><infon key="type">paragraph</infon><offset>37582</offset><text>Experiments presented here reveal a link between two  NE-embedded organelles that are functionally quite different. Little is known about how NPCs and SPBs are correctly assembled into the NE, but these processes are  expected to be complex, requiring the organelles to be oriented correctly and possibly to be dependent on the function and/or assembly of each other. The localization of  Ndc1p to both of these organelles suggests a common  mechanism for the assembly of NPCs and SPBs into the NE.</text></passage><passage><infon key="type">title_1</infon><offset>38081</offset><text>Figures and Tables</text></passage><passage><infon key="type">table_caption</infon><offset>38100</offset><text>Yeast Strain List </text></passage><passage><infon key="type">table</infon><offset>38119</offset><text>   Strain*  Genotype   ProA5-4d/4a  MATa/MATalpha NDC1-ProA-HIS3-URA3/NDC1-ProA-HIS3-URA3 ura3-52/ura3-52 his3Delta200/his3Delta200 trp1-1/trp1-1     leu2-3,112/leu2-3,112 lys2-801/lys2-801  ProA5-4d  MATaNDC1-ProA-HIS3-URA3 ura3-52 his3Delta200 trp1-1 leu2-3,112 lys2-801  HC26-15a/1a  MATa/MATalpha NDC1-ProA-HIS3-URA3/NDC1-ProA-HIS3-URA3 nup49-1::URA3/nup49-1::URA3 nup49DeltaGFLG::GFP-     S65T-TRP1/nup49DeltaGFLG::GFP-S65T-TRP1 ura3/ura3 his3/his3 trp1-1/trp1-1 leu2-3,112/leu2-3,112  HC27-16d/16b  MATa/MATalpha nup133Delta::HIS3/nup133Delta::HIS3 NDC1-ProA-HIS3-URA3/NDC1 nup49-1::URA3/nup49-1::URA3     nup49DeltaGFLG::GFP-S65T-TRP1/nup49DeltaGFLG::GFP-S65T-TRP1 ura3/ura3 his3/his3 trp1-1/trp1-1 leu2-3,112/leu2-3,112  HC22-2b/1c  MATa/MATalpha NDC1-ProA-HIS3-URA3/NDC1-ProA-HIS3-URA3 spc42Delta::LEU2/spc42Delta::LEU2     TRP1::SPC42-GFP(3x)/TRP1::SPC42-GFP(3x) ura3/ura3 his3/his3 trp1-1/trp1-1 leu2-3,112/leu2-3,112 ade2-1/ade2-1  HC23-11d/16a  MATa/MATalpha NIC96-ProA-HIS3-URA3/NIC96-ProA-HIS3-URA3 spc42Delta::LEU2/spc42Delta::LEU2 TRP1::SPC42-     GFP(3x)/TRP1::SPC42-GFP(3x) ura3/ura3 his3/his3 trp1-1/trp1-1 leu2-3,112/leu2-3,112 ade2-1/ade2-1  HC14-10c(1198)/HC29-6b  MATa/MATalpha ndc1Delta::KANMX/ndc1Delta::HIS3 ura3-52/ura3-52 his3Delta200/his3Delta200 trp1Delta63/TRP1 leu2Delta1/leu2Delta1 LYS2/     lys2-801 ade2Delta426/ade2Delta426 ade3Delta/ade3Delta (pRS315-NDC1-GFP)  HC10-42b/42c  MATa/MATalpha ura3-52/ura3-52 ade2/ade2 his4-539/HIS4  HC10-42a/42d  MATa/MATalpha ndc1-1/ndc1-1 ura3-52/ura3-52 ade2/ade2 his4-539/HIS4  HC13-1c  MATalpha NIC96-ProA-HIS3-URA3 ura3-52 his3Delta200 trp1-1 leu2-3,112 lys2-801  Wx340-3c  MATalpha ndc1-1 tub2-403 ura3-52 his4-539 lys2-801  Wx340-3d  MATalpha tub2-403 ura3-52 his4-539  HC14-10c  MATaura3-52 his3Delta200 trp1Delta63 leu2Delta1 ade2Delta426 ade3Delta  HC5-31c  MATandc1-1 ura3-52 his3Delta200 trp1Delta63 leu2Delta1 ade2Delta426 ade3Delta  HC5-31c(1166)  MATandc1-1 pom152Delta::HIS3 ura3-52 his3Delta200 trp1Delta63 leu2Delta1 ade2Delta426 ade3Delta</text></passage><passage><infon key="type">table_footnote</infon><offset>40004</offset><text> All strains were constructed as a part of this study.   </text></passage><passage><infon key="type">fig_caption</infon><offset>40065</offset><text>Ndc1p-ProA localizes to  NPCs in whole cells. (A) Indirect IF  deconvolution microscopy (see Materials and Methods) of yeast strain  ProA5-4d/4a (Table I). Ndc1p-ProA  was indirectly labeled using an FITC-conjugated secondary antibody, and  DNA was visualized using 4',6-diamidino-2-phenylindole. (B) Indirect IF  deconvolution microscopy of yeast  strain HC26-15a/1a (Table I) containing Ndc1p-ProA and Nup49p-GFP  (Bucci and Wente, 1997). Ndc1p-ProA  was indirectly labeled using a Texas  red-conjugated secondary antibody,  and Nup49p-GFP autofluorescence  was detected using FITC filters. The  combined image shows both Texas red  (red) and GFP (green) signal (overlap =  yellow). (C) Indirect IF deconvolution  microscopy of Ndc1p-ProA localization and Nup49p-GFP localization in  the presence of the nup133 null allele,  which causes NPCs to cluster (HC27-16d/16b; Table I) (Doye et al., 1994;  Pemberton et al., 1995). Ndc1p-ProA  and Nup49p-GFP were detected as in  B. The combined image shows both  Texas red (red) and GFP (green) signal  (overlap = yellow).</text></passage><passage><infon key="type">fig_caption</infon><offset>41137</offset><text>Biochemical analysis of Ndc1p-ProA. (A) Biochemical  fractionation was carried out using a yeast strain that contains  Ndc1p-ProA (ProA5-4d; Table I) (Rout and Kilmartin, 1990;  Rout and Kilmartin, 1991; Rout and Blobel, 1993). Fractions containing nuclei, crude NPCs, SPBs, and enriched NPCs (E-NPCs)  are labeled. Individual fractions were analyzed by SDS-PAGE  and immunoblotted using antibodies to detect Pom152p, Ndc1p-ProA, or Spc110p. Pom152p is a NPC component (Wozniak et al.,  1994), and Spc110p is a SPB component (Rout and Kilmartin,  1990; Kilmartin et al., 1993). Cell equivalents loaded were 1n for  the left panels and 10n for the middle and right panels. (B) NEs  were isolated from the Ndc1p-ProA yeast strain (ProA5-4d; Table I) using a previously described technique (Strambio-de-Castillia et al., 1995). Cell equivalents loaded were 1n for the left  panels and 3n for the right panels. (C) Samples from the NE fraction were extracted with carbonate followed by centrifugation to  determine whether Ndc1p-ProA was found in the supernatant  (S) or in the membrane-containing pellet (P) (see Materials and  Methods). Samples were analyzed by SDS-PAGE and immunoblotted using antibodies to detect Ndc1p-ProA, Pom152p, or  Nup57p.</text></passage><passage><infon key="type">fig_caption</infon><offset>42384</offset><text>Ndc1p-ProA localizes to SPBs in whole cells.  (A) Indirect IF deconvolution microscopy of cells  (HC22-2b/1c; Table I) containing Ndc1p-ProA and  Spc42p-GFP. Ndc1p-ProA  was detected using a Texas  red-conjugated secondary  antibody, and Spc42p-GFP  was detected by means of  GFP autofluorescence using  FITC filters (see Materials  and Methods). The combined  image shows both Texas red  (red) and FITC (green) signal  (overlap = yellow). (B) Indirect IF deconvolution microscopy of cells containing  Nic96p-ProA and Spc42p-GFP (HC23-11d/16a; Table  I) serves as a negative control  for A. Samples were prepared as in A. The combined  image shows both Texas red  (red) and FITC signal  (green). (C) Indirect IF deconvolution microscopy to  examine the colocalization  of Ndc1p-ProA with Nup49p-GFP in a nup133 null strain  that exhibits NPC clustering  (HC27-16d/16b; Table I).  Ndc1p-ProA was detected as  in A, and Nup49p-GFP was  detected using FITC filters.  The combined image shows  both Texas red (red) and  FITC (green) signals (overlap = yellow). Shown here  are examples of cells that  contain either one or two extra spots of Ndc1p-ProA  staining that do not colocalize with Nup49p-GFP signal.</text></passage><passage><infon key="type">fig_caption</infon><offset>43592</offset><text>Ndc1p-GFP localizes to the membrane interacting regions of NPCs and  SPBs. Ndc1p-GFP localization was examined using a  diploid strain that is homozygous for the ndc1 null allele  and is transformed with a  centromeric plasmid containing NDC1-GFP [HC14-10c  (1198)/HC29-6b; Table I].  Ndc1p-GFP localization was  observed in living cells using  deconvolution fluorescence  microscopy (A). Additionally, this localization was examined using immunoelectron microscopy of whole  cells (see Materials and  Methods). Immunogold labeling, corresponding to  Ndc1p-GFP, was detected at  the periphery of NPCs (B)  and at the edges of the central plaque region of SPBs  (C); these are the regions of  NPCs and SPBs that interact  with the NE.</text><annotation id="5"><infon key="type">ExperimentalMethod</infon><infon key="PSIMI">0416</infon><location offset="43877" length="226"/><text>Ndc1p-GFP localization was  observed in living cells using  deconvolution fluorescence  microscopy (A). Additionally, this localization was examined using immunoelectron microscopy of whole  cells (see Materials and  Methods).</text></annotation></passage><passage><infon key="type">fig_caption</infon><offset>44326</offset><text>ndc1-1 strains do  not exhibit defects in NPC  assembly. An assay to examine NPC assembly was carried out using either wild-type (WT) or ndc1-1 cells  (HC10-42a/42d and HC10-42b/42c, respectively; Table  I) transformed with a plasmid containing NIC96-ProA  expressed under the control  of a galactose-inducible promoter (pGAL-NIC96-ProA).  Asynchronous cultures were  shifted to 13.5 C for 18 h  (18h); at this point, the  ndc1-1 culture contained  80% large budded cells.  NIC96-ProA expression was  induced for 12 h (30h), followed by a glucose chase for  4 h to determine whether  Nic96p-ProA was properly  assembled into NPCs (34h).  Nic96p-ProA localization  was visualized using indirect  IF microscopy with an FITC-conjugated secondary antibody (see Materials and  Methods).</text></passage><passage><infon key="type">fig_caption</infon><offset>45109</offset><text>Deletion of POM152 suppresses the ndc1-1 SPB duplication defect in the first cell cycle. (A) ndc1-1 or ndc1-1, pom152  null double mutant strains [HC5-31c and HC5-31c(1166), respectively; Table I] containing pURA3-NDC1 (pALR10-NDC1, see  Materials and Methods) or lacking pURA3-NDC1 were transformed with either pRS315 (pLEU2) or pPM1-HA (pLEU2-POM152) and were streaked to a 15 C Leu- plate. All strains  containing pURA3-NDC1 were able to grow at the nonpermissive temperature. The ndc1-1 strain lacking pURA3-NDC1 failed  to grow. However, the ndc1-1, pom152 null double mutant strain  grew at 15 C when it contained the pLEU2 vector (*). When a  plasmid-borne copy of POM152 (pLEU2-POM152) was reintroduced, the double mutant strain exhibited the ndc1-1 cold-sensitive phenotype (**). (B) DNA content of wild-type (WT), ndc1-1,  and ndc1-1, pom152 null double mutant (ndc1-1, pom152Delta)  strains [HC14-10c, HC5-31c, and HC5-31c(1166), respectively;  Table I]. Asynchronous cultures (0:00, dashed lines) were arrested in G1 (1c DNA content; G2 and M cells have 2c DNA content) by treatment with alpha-factor at the permissive temperature (0:00, bold line) (see Materials and Methods). Cells were  released from this arrest into media at the nonpermissive temperature for ndc1-1 for 19.5 h (19:30).</text></passage><passage><infon key="type">title</infon><offset>46413</offset><text>Abbreviations used in this paper</text></passage><passage><infon key="type">paragraph</infon><offset>46446</offset><text>GFP</text></passage><passage><infon key="type">paragraph</infon><offset>46450</offset><text>green fluorescent protein</text></passage><passage><infon key="type">paragraph</infon><offset>46476</offset><text>IF</text></passage><passage><infon key="type">paragraph</infon><offset>46479</offset><text>immunofluorescence</text></passage><passage><infon key="type">paragraph</infon><offset>46498</offset><text>NE</text></passage><passage><infon key="type">paragraph</infon><offset>46501</offset><text>nuclear envelope</text></passage><passage><infon key="type">paragraph</infon><offset>46518</offset><text>NLS</text></passage><passage><infon key="type">paragraph</infon><offset>46522</offset><text>nuclear localization signal</text></passage><passage><infon key="type">paragraph</infon><offset>46550</offset><text>NPC</text></passage><passage><infon key="type">paragraph</infon><offset>46554</offset><text>nuclear pore complex</text></passage><passage><infon key="type">paragraph</infon><offset>46575</offset><text>ProA</text></passage><passage><infon key="type">paragraph</infon><offset>46580</offset><text>protein A</text></passage><passage><infon key="type">paragraph</infon><offset>46590</offset><text>SPB</text></passage><passage><infon key="type">paragraph</infon><offset>46594</offset><text>spindle pole  body</text></passage><passage><infon key="type">title</infon><offset>46613</offset><text>References</text></passage><passage><infon key="volume">131</infon><infon key="name_2">surname:Rout;given-names:MP</infon><infon key="name_1">surname:Blobel;given-names:G</infon><infon key="name_0">surname:Aitchison;given-names:JD</infon><infon key="fpage">1659</infon><infon key="pub-id_pmid">8557736</infon><infon key="year">1995a</infon><infon key="source">J Cell Biol</infon><infon key="type">ref</infon><infon key="lpage">1675</infon><offset>46624</offset><text>Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport</text></passage><passage><infon key="name_3">surname:Blobel;given-names:G</infon><infon key="volume">131</infon><infon key="name_2">surname:Marelli;given-names:M</infon><infon key="name_1">surname:Rout;given-names:MP</infon><infon key="name_0">surname:Aitchison;given-names:JD</infon><infon key="fpage">1133</infon><infon key="pub-id_pmid">8522578</infon><infon key="year">1995b</infon><infon key="source">J Cell Biol</infon><infon key="type">ref</infon><infon key="lpage">1148</infon><infon key="name_4">surname:Wozniak;given-names:RW</infon><offset>46702</offset><text>Two novel related yeast nucleoporins Nup170p and Nup157p: complementation with the vertebrate homologue Nup155p and functional interactions with the yeast nuclear pore-membrane protein Pom152p</text></passage><passage><infon key="citation">Ausubel, J.D., R. 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