POM152 Is an Integral of the Pore Membrane Domain of the Yeast

Richard W. Wozniak, Gfinter Blobel, and Michael P. Rout Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York 10021

Abstract, We have identified a concanavalin A-reac- COOH-terminal region (1,142 residues) positioned on tive glycoprotein of 150 kD that coenriches with iso- the pore side and cisternal side of the pore membrane, lated yeast complexes. Molecular cloning respectively. The proposed cisternally exposed domain and sequencing of this protein revealed a single canon- contains eight repetitive motifs of ~ 24 residues. Sur- ical transmembrane segment. Epitope tagging and lo- prisingly, POM152 deletion mutants were viable and calization by both immunofluorescence and immuno- their growth rate was indistinguishable from that of electron microscopy confirmed that it is a pore wild-type cells at temperatures between 17 and 37°C. membrane protein. The protein was termed POM152 However, overproduction of POM152 inhibited cell

(for pore membrane protein of 152 kD) on the basis growth. When expressed in mouse 3T3 cells, POM152 Downloaded from of its location and cDNA-deduced molecular mass. was found to be localized to the pore membrane, sug- POM152 is likely to be a type II membrane protein gesting a conserved sorting pathway between yeast and with its NHz-terminal region (175 residues) and its mammals. www.jcb.org

UCLEAR pore complexes (NPCs) 1 are macromo- review see Akey, 1992). Conversely, the pore-exposed bulk lecular assemblies that serve to regulate nucleo- of POM121 is most likely an integral part of the pore side components of the NPC as it shares a repetitive pentapeptide N cytoplasmic communication (for review see Forbes, on April 12, 2006 1992). They reside in circular openings (nuclear pores) motif (Hallberg et ai., 1993) that has also been identified in across the nuclear envelope (NE) (for review see Franke, some NPC (Davis and Fink, 1990; Nehrbass et al., 1974; Gerace and Burke, 1988). The nuclear pore mem- 1990; Starr et al., 1990; Sukegawa and Blobel, 1993). branes are morphologically and biochemically distinct do- One of the most likely functions of these pore membrane mains of the NE that border the nuclear pores. So far, two proteins is the anchoring of the NPC in the nuclear pore integral membrane proteins have been identified in higher (Gerace et al., 1982; Wozniak et al., 1989; Hallberg et al., eukaryotes that are specifically located in the pore mem- 1993). Such proteins may also play a role in regulating brane domain, namely gp210 (Gerace et al., 1982; Wozniak nucleocytoplasmic traffic through the NPC. Greber and Ger- et al., 1989) and POM121 (Hallberg et al., 1993). Both ace (1992) have shown that a monoclonal antibody against membrane proteins have a single transmembrane segment. the cisternal domain of gp210 can reduce the rate of protein However, whereas most of the mass of gp210 is located on import into the nucleus. Furthermore, integral pore mem- the cisternal side of the pore membrane 0Nozniak et al., brane proteins may be involved in the circumscribed fusion 1989; Greber et al., 1990), the bulk of POM121 faces the of the inner and outer nuclear membrane to form new nu- pore side of the pore membrane (Hallberg et al., 1993). clear pores (Maul, 1977; Wozniak et al., 1989). These fu- Gp210 could contribute either to the "lumenal" spokes or the sion processes could also be involved in the elimination of radial arms that have been identified in ultrastructural analy- nuclear pores by restoring the double membrane. sis (Hinshaw et al., 1992; Akey and Radermacher, 1993; for Integral proteins of the pore membrane domain have not previously been identified in yeast. However the recent isola- tion of NPCs from yeast has allowed the identification of a predominant, constituent concanavalin A (ConA)-binding Address all correspondenceto R. W. Wozniak. Dr. Wozniak's current ad- glycoprotein. We have determined that this protein is an inte- dress is the Department of Anatomy and Cell Biology, Universityof Al- gral protein of the pore membrane domain. The protein was berta, Edmonton, Alberta, Canada T6G 2H7. termed POM152 on the basis of its cDNA-deduced primary structure and calculated molecular mass of 151,670 daltons. 1. Abbreviations used in this paper: Con A, concanavalinA; HA, hemag- glutinin; NE, nuclear envelope; NPC, nuclear pore complex; POM, pore Unexpectedly, deletion mutants of the POM152 are via- membrane protein; SDS-HA, SDS-hydroxylapatite;SM-URA, synthetic ble. When expressed in mouse 3T3 cells the yeast protein medium lacking uracil. specifically localized to the mammalian pore membrane.

© The RockefellerUniversity Press, 0021-9525/94/04/31/12 $2.00 The Journalof Cell Biology,Volume 125, Number 1, April 199431--42 31 Materials and Methods bonate, pH 11.5, was added to the suspended NEs and the sample was in- cubated for 15 min. Extracted proteins were separated from the NE mem- braue by centrifugation at 436000 g for 30 rain in a TLA 100.2 rotor Strains and Media (Beckman Instruments Inc., Palo Alto, CA). The supernatant was collected The yeast strains used in this study are listed in Table I. They were grown and proteins were precipitated with 10% TCA. This precipitate, the mem- as previously described (Sherman et ai., 1986) in either YPD (1% yeast ex- brane pellet, and the starting NE fraction were solubilized in SDS-sample tract, 2% bactopeptone, and 2% glucose)or synthetic minimal media (SM) buffer in preparation for SDS-PAGE. The gels were either stained with supplemented with the appropriate amino acids and either 2% glucose or Coomassie blue or the polypeptides electrophoretically transferred to 2% gaiactose. Standard procedures for yeast genetic manipulations were as nitrocellulose, probed with 14C-labeled Con A (Sigma Chemical Co., St. described in Sherman et ai. (1986). Transformations of yeast using lithium Louis, MO), and visualized by fluorography as previously described (Woz- acetate were performed as described in Ito et al. (1983). niak et ai., 1989).

Fractionation of Yeast NPC Proteins Isolation and Sequencing of the Gene Encodingp150 Approximately 5 mg of enriched yeast nuclear pore complexes, isolated The sequence of a peptide fragment of p150 corresponding to amino acid from Saccharomyces uvarum as described by Rout and Blobel (1993), were residues 332-353 was used to determine the exact cDNA sequence of p150 solubilized in 2% SDS, 100 mM sodium phosphate buffer, pH 6.8, 100 mM in this region using the PCR procedures (Lee et al., 1988). SyntheSis, isola- DTT, and 0.5 mM PMSE Polypeptides were fiactionated by SDS-hydroxyl- tion, subeloning, and sequencing of the PCR products were performed as apatite (SDS-HA) chromatography as previously described (Courvalin et previously described (Radu et al., 1993) with the following moditicatious. al., 1990) except that the linear elution gradient was 0.2-0.75 M sodium The two partially degeuerate oligonucleotides were synthesized corm- phosphate, pH 6.8, containlns 0.1% SDS and 1 mM DTT. For SDS-PAGE sponding to the sense sequence of amino acid residues 332-337 and the anti- analysis, aiiquots from fractions were diluted twofold in SDS-sample buffer sense sequence of amino acids 349--353. The template for PCR was Sac- and loaded directly onto the gel. charomyces cerevisiae genomic DNA (0.5 t~g per reaction) and the anneal- Further separation of polypeptides from SDS-HA fractions containing ing temperature was adjusted to 50°C. a Con A-binding protein of an estimated mass of 150 kD was achieved by On the basis of the sequence of the PCR product a 41-mer oligonucleo- reverse-phase HPLC. Fractions from the SDS-HA eluate containing p150 tide complementary to the sense strand was synthesized. This oligonucleo- were pooled and directly loaded onto an Aquapore butyl (C-4) column (100 tide was end labeled with 3,-[32P] ATP (New England Nuclear, Boston, × 10 ram, Brownlee Labs, Applied Biosystems Inc., Foster City, CA) MA) using T4 pulynucleotide kinase (New England Biolabs, Beverly, MA) equilibrated with 60% formic acid. After a 5-rain linear increase to 6.6% and used to screen a S. cerevisiae genomic DNA library in lambda DASH acetonitrile in 60% formic acid, the column was eluted with a 1-h linear (,~450,000 pfus) (Stratngeue Cloning Systems, La Jolla, CA). Phage lifts Downloaded from gradient of 6.6-33% acetonitrile in 60% formic acid. In preparation for were performed as described (Benton and Davis, 1977). Prehybridization, electrophoresis, aiiquots of the eluted fractions were dried in a Speed Vac hybridization, and washing of filters were conducted as described (Radu et Concentrator (Savant Instruments Inc., Hicksville, NY). Dried pellets were ai., 1993). Five overlapping clones were isolated that represent a 7.9-kb solubilized in SDS-sample buffer, heated at 65°C for 20 rain, and then ana- fragment of genornic DNA containing the geue encoding p150 (shown sche- lyzed by SDS-PAGE. matically in Fig. 6 A). Inserts from these clones were excised with SaiI and FOr cleavage and sequencing of p150, HPLC fractions containing this subeloned into pBluescript II SK(-) (Stratageue Cloning Systems). protein were pooled, prepared for SDS-FAGE as above, and separated on Double-stranded sequencing of plasmid DNA (Mierendoff and Pfeffer, a 6% polyacrylamide gel. Polypeptides were then electrophoretically trans- 1987) was performed with synthetic oligonucleotide primers using Se- www.jcb.org ferred to polyvinyldiene difluoride membrane and visualized with 0.1% quenase (United States Biochemical Corp., Cleveland, OH). For determin- Fonceau red in 1% acetic acid. p150 was excised and cleaved with endopep- ing the sequence across a single internal SaiI site, lambda DNA (10 t~g) was tidase Lys-C as described (Fernandez et al., 1992) and several internal pep- sequenced directly using the same procedure. A 5,482-bp fragment, which tides were subjected to NH2-terminai sequence analysis.

was bidirectionally sequenced, containing the p150 (now termed POM152) on April 12, 2006 open reading frame is shown in Fig. 4. Sodium Carbonate Extraction of Yeast NEs For expression purposes, a cDNA bordered by BamHI sites and contain- ing the complete POMI52 geue was assembled in pBluescript II SK(-) from Yeast NEs were isolated from S. uvarum using the procedure of Kilmartin three restriction fragments isolated from separate lambda clones: a 1.3-kb and Fogg (1982). All manipulations were done at 4"C. 0.25 mg of NE pro- BamHI/BgllI fragment from the 5' end of the geue, a 2.8-kb BglII/SalI inter- teins were suspended in 0.5 nd of 10 mM bisTris, pH 6.5, 0.1 mM MgC12, nal fragment, and a 2.6-kb SalI/BamHI 3' fragment (see schematic, Fig. 6 1 mM DTT, and 0.1 mM PMSF. An equal volume of 0.2 M sodium car- A). The resulting plasmid is termed pBPMI.

Table L Yeast Strain Genotype Strain Genotype Derivation W303 Mata/Matcx ade2-l/ade2-1 ura3-1/ura3-1 his3-11,15/his3-11,15 trpl-1/trpl-1 leu2-3,112/leu2-3,112 canl-lOO/canl-lO0 PMY1 Mata/Mata ade2-1/ade2-1 ura3-1/ura3-1 Integrative transformation of his3-11,15/his3-11,15 trpl-1/trpl-1 W303 with BamHI fragment of leu2-3,112/leu2-3,l12 canl-lOO/canl-lO0 pPM1-HIS pom152-2 : :H1S3/+ PMY17 Mata ade2-1 ura3-1 his3-11,15 trpl-1 Segregant of sporulated PMY1 1eu2-3,112 canl-lO0 pom152-2: :HlS3 PMY17-HA Mata ade2-1 ura3-1 his3-11,15 trpl-1 Transformation of PMY17 Ieu2-3,112 canl-lO0 pom152-2::HlS3 with pPM1-HA pPM1-HA(LEU2) PMGal 1 Mata/Mata ade2-1/ade2-1 ura3-1/ura3-1 Transformation of W303 his3-11,15/his3-11,15 trpl-1/trpl-I with pPMGal leu2-3,112/leu2-3,112 canl-lOO/canl-lO0 pPMGaI(URA3)

The Journal of Cell Biology, Volume 125, 1994 32 Epitope Tagging of POM152 for the Lowicryl sections except the antibody was diluted 1:5 in PBS con- taining 0.5% BSA. The grids were stained and processed according to For immunolocalization studies of POM152, an epitope tag encoding two Griffiths et al. (1983). tandemly repeated 12-amino acid residue peptides, 10 from the influenza For the Con A-gold staining of isolated NPCs, samples of the enriched virus hemngglutinin (HA) molecule (Wilson ct al., 1984; Field et al., 1988) NPC fraction were prepared for negative stain electron microscopy as de- plus two flanking glycines added as spacers, was inserted into POM152 after scribed (Rout and Elobel, 1993). However, the final uranyl acetate staining amino acid residue 293 (see Fig. 4). This was accomplished by synthesizing step was replaced with a wash of the grids in Con A blot buffer (Evans et two complementary oligonucleotides, yHA-1 and yHA-2, with the following al., 1986), followed by an overnight incubation at room temperature in a sequence. 1:300 dilution of 20-nm colloidal gold-labeled Con A (Sigma Chemical Co.) in Con A blot buffer. Control incubations also contained 0.5 M methyl 5'gat ctt ggt tac cca tac sac gtc cca sat tac gct agc ggt 3' yHA-I c~-D-mannopyranoside. After extensive washing in Con A blot buffer, the 3' aa CCa atg ggt atg ctg ca8 ggt cta at8 cga tcg cca cta g 5' yHA-2 grids were fixed in 1.25 % glutaraidehyde in the same buffer (30 min, room G Y P Y D V P D Y A S G amino acid residues temperature) and negatively stained with 4% uranyl acetate. All images were recorded on Kodak electron microscope film. An excess of this oligonucleotide pair was llgated into a unique BglII site (nucleotide 1621) of the POM152 gene in pBPM1 and clones containing two tandemiy repeated inserts with the proper orientation were identified Expression of poml52: :HA in Mouse NIH 3T3 Cells by DNA sequencing. The tagged POM/52 gene (pom152-2::HA) within a A full-length cDNA containing the coding region ofpoml52-2::HA was as- BamHI/BamHI fragment was subcloned into pRS315 to produce the plas- sembled from two PCR products, a 5' segment of 2,420 bp and a 3' segment mid pPM1-HA. of 2,165 bp. The 5' product was synthesized using a sense primer consisting of a 5' XbaI site followed by nucleotides 842-859 of the POMI52 gone (in- Disruption of the POMI52 Gene cluding the initiation codon) and an anti-sense primer corresponding to nucleotides 3,241-3,257. The 3' segment was synthesized using a sense Deletion and disruption of the POM152 gone was performed by integrative primer encoding nucleotides 2,719-2,742 and an anti-sense primer encod- transformation using the procedure of Bothstein (1991). The construct used ing a 5' mHI site followed by nucleotides 4,865-4,882 (including the ter- for transformation (pPM1-HIS) was assembled in pBluescript 11 SK(-) and mination codon). The template used for both reactions was the pPM1-HA consisted of a BamHI/XhoI DNA fragment containing the HIS3 selectable plasmid containing pom152-2::HA. Both amplification products overlap marker isolated from the vector pJJ217 (Jones and Parkash, 1990) flanked across a region of the POM152 gene that contains a unique Eco0109I site by genomic DNA sufficient for recombination at the POM152 locus. Flank- (at nucleotide 3,088). The two products were cleaved with Eco0109I and ei- ing the HIS3 gene on the 5' side was a 525-bp amplification product extend- ther XbaI or BamHI and together assembled in the multiple cloning site of ing from a BamHI site 5' of the POM152 gene to an MboI site at nucleotide the mammalian expression vector pSVL (Pharmacia LKB Biotechnology, Downloaded from 915. The 3' segment was derived from a 2.6-kb SaiI/BamHI restriction frag- Piscataway, NJ). Thepom152::HA/pSVL plasmid was introduced into 3T3 ment containing the 3' end of the POM152 gene and sequences downstream cells by direct microinjectiun (Capecchi, 1980). Expression was detected to a BamHI site (see Fig. 6 A for map of this construct). 21 h after injection by indirect immunofluorescence using the 12CA5 mAb A BamHI/BamHI fragment of pPMI-His was transformed into the S. and Texas red-labeled goat anti-mouse IgG (Cappel Laboratories, Organon cerevisiae diploid strain W303 and His+ transformants were isolated. Het- Teknika Corp., Durham, NC) as described (Wozniak and Elobel, 1992). erozygous diploids carrying the integrated pom152-1::HIS3 disrupted gene

and the wild-type allele were identified by Southern blotting. Cells (PMY1) www.jcb.org were sporulated and tetrad analysis was performed. The expected 2:2 segre- GALl-directed Overexpression of POM152 gation of the His+ marker was observed. The absence of the wild-type POM152 gene in the His+ haploids was confirmed by Southern analysis of The PCR-derivedpom152::HA cDNA was subcloned into the yeast expres- BamHI-digested genomic DNA (5 t~g) isolated from each of four segre- sion plasmid pRS426 (Christianson et al., 1992) 3' of the GALl promoter

gants. Probing was conducted using the 5' PCR product described above (la- to produce the plasmid pPMGal. This plasmid and pRS426 were indepen- on April 12, 2006 beled with 32p by random priming) under conditions described for library dently transformed into the diploid strain W303 and viable Lira+ transfor- screening. mants were selected. Single colony transformants were propagated on syn- thetic medium lacking uracil and containing 2% glucose (SM-URA Indirect Immunofluorescence and glucose). To examine the effect of POM152 overexpression on cell growth, cells were transferred to SM-URA containing 2% galactose (SM-URA Immunoelectron Microscopy galactose) or SM-URA glucose media and incubated for 12 h at 30°C Cells The yeast haploid strain PMY17-HA containing the tagged POM152 gene were then s~ onto appropriate plates containing SM-URA galactose (pom152-2::HA) was used for both immunottuorescence and immunoelec- or SM-URA glucose, incubated for 48 h at 30°C, and photographed. For tron microscopy. Cells from early log phase cultures were prepared for in- immunnfluorescence studies, cells were grown to early log phase in SM- direct immunnfluorescence using the procedure of Kilmartin and Adams URA glucose. Cells were then washed 3x with water and 2× with SM- (1984) with the modifications of Wente et al. (1992). Probing for the URA galactose. Washed cells were subsequently incubated in SM-URA epitope-tagged POM152 with an mAb specific for the HA peptide (12CA5; gaiactose for 10 h at 30°C. Indirect immunofluorescence was conducted Berkeley Antibody Co., Richmond CA) and detection with FITC labeled using the 12CA5 antibody as described above. goat anti-mouse IgG (Cappel Laboratories, Organon Teknika Corp., Dur- ham, NC) was performed as previously described (Wente et ai., 1992). Photographs were taken through a 100× objective onto Kodak T-Max 400 film (Eastman Kodak Co., Rochester, NY) and processed at 1600 ASA. Results FOr immunoelectron microscopy, spheroplasts (Byers and Goetsch, 1991) from early log phase cultures of PMY17-HA cells were washed and Isolated Yeast Nuclear Pore Complexes Contain a fixed essentially as described in Wente et al. 0992). Spheroplasts were then pelleted, dehydrated in graded alcohol, and embedded in Lowicryl accord- 150-kD Con A-binding Protein ing to the manufacturer's instructions (Electron Microscopy Sciences, Fort Recently, an enrichment procedure has been developed for Washington, PA). Ultrathin sections were collected on Formvar carbon- the isolation of NPCs from the yeast Saccharomyces (Rout coated nickel grids. The sections were probed with mAb 12CA5 (see above) diluted 1:2 in PBS for 2-3 h at room temperature. After washing with PBS, and Blobel, 1993). Analysis of the fractions from the enrich- the sections were incubated with goat anti-mouse IgG bound to 10 nm gold ment procedure by SDS-PAGE and immunoblotting con- (Amersham Corp., Arlington Heights, IL). Sections were stained with ura- firmed the coenrichment of known NPC proteins with the nyl acetate and viewed in a JEOL 100CX electron microscope operated at isolated NPCs (Rout and Blobel, 1993). When parallel blots 80 IN. Alternatively, fixed cells were pelleted and embedded in 10% gelatin. were performed with ~4C-Con A (Fig. 1), a single band of Samples were then refixed for 1 h at 4°C with the same fixative. The pellet was infused with 2.3 M sucrose, frozen in liquid nitrogen, and then cryosec- '~150 kD (p150) (Fig. 1, compare lanes 7, 10, 16, and 21) was tioned (Tokuyasu, 1973). The immunolabeling was the same as described similarly found to coenrich with the NPCs, coincident with a

Wozniak et al. POM 152, a Yeast Pore Membrane Protein 33 Downloaded from

Figure 2. p150 is an integral membrane protein of yeast nuclear envelopes. Yeast NEs (lane NE) were extracted with 0.1 M sodium carbonate, pH 11.5, and then sedimented by centrifugation to yield www.jcb.org a supernatant fraction (lane s) containing peripheral proteins and a membrane pellet containing integral proteins (lane p). Proteins of these two fractions, as well as of enriched NPCs (NPC), and of

a fraction from the C-4 HPLC eluate containing p150 (Fr.68) (see on April 12, 2006 Fig. 3 B) were separated by SDS-PAGE and either stained with Coomassie blue (panel A) or transferred to nitrocellulose and probed with ~4C-Con A (B). Coomassie blue staining revealed a protein of similar mobility as p150 (A, lane Fr.68) in the NPCs, the NE, and the extracted membrane pellet fractions (A, lanes NPC, Figure 1. Coenrichment of a Con A-binding protein of ~150 kD NE, and p). Consistent with this, t4C-Con A revealed that p150 with yeast NPCs. Five separate sucrose gradients that sequentially was present in the NE fraction (B, lane NE) and that it remained led to enriched NPCs (see Rout and Blobel, 1993) were analyzed associated with the extracted membrane fraction (B, lane p). No by SDS-PAGE and 14C-Con A blotting to identify glyeoproteins Con-A binding protein of similar mobility is visible in the superna- that cofraetionate with NPCs. Gradients and gradient fractions tant (B, lane s). Molecular mass markers in kilodaltons are shown were prepared exactly as described in Rout and Blobel (1993). to the fight. Coomassie blue staining of similar fractions can be seen in this ref- erence. Fractions from each gradient are grouped as indicated by the lines beneath the gel with the first gradient on the left and the To determine whether p150 is in fact an integral membrane last on the fight. The numbers below the lines represent the equiva- protein, we extracted isolated yeast NEs with 0.1 M sodium lents (in arbitrary n units) loaded per lane relative to n units of start- carbonate, pH 11.5, and by subsequent centrifugation sepa- ing ceils. Spheroplasts and subsequent fractions containing NPCs rated peripheral from integral membrane proteins in a super- starting with crude nuclei (lane 3) and continuing to highly en- natant (s) and pellet fraction (p), respectively (Fig. 2 A). In- riched NPCs (lane 21) are indicated above the gel. A single Con deed, a Coomassie staining band of '~150 kD was present A-binding protein of 150 kD (arrowhead, lane 21 ) is visible coen- riching with NPCs. Molecular mass markers in kilodaltons are in the NE and resisted high pH extraction (Fig. 2 A, compare shown to the left of the gel. lanes p and NE). This membrane-integrated protein comi- grated with the p150 protein present in isolated NPCs and in a highly enriched fraction of p150 (Fig. 2 A, lanes NPC prominent Coomassie staining band of the same relative mo- and Fr.68: for the origin of Fr.68 see Fig. 3 B). Probing of bility (see Rout and Blobel, 1993 and Fig. 2 A, lane NPC). the SDS-PAGE-separated proteins with 14C-Con A showed The Con A reactivity implied that this protein was an integral that the Con A-reactive p150 in the isolated NPCs and the membrane component containing N-linked carbohydrates; NPC subfraction (Fig. 2 B, lanes NPC and Fr.68) comigrated thus its coenrichment with the isolated NPCs strongly sug- with a Con A-reactive protein in the NE (Fig. 2 B, lane NE) gested it as a candidate pore membrane protein. that resisted extraction by alkali (Fig. 2 B, lane p). No Con

The Journal of Cell Biology, Volume 125, 1994 34 A-binding proteins of similar mass were visible in the high pH extract (Fig. 2 B, lane s). These results suggested that the Con A-reactive p150 of the NPC fraction is an integral pro- tein of the pore membrane. To purify p150, enriched NPCs were solubilized in SDS and the proteins separated by SDS-HA chromatography (Fig. 3 A). In column fractions analyzed by SDS-PAGE, p150 was visible in fractions 34-38 both by Coomassie stain- ing (Fig. 3 A, arrowhead) and by 14C-Con A blotting (data not shown). Further separation of polypeptides in the pl50- containing fractions was accomplished by reverse phase HPLC (Fig. 3 B). p150 was purified from HPLC fractions by preparative SDS-PAGE, transferred to PVDF membrane, and subjected to endoproteolytic cleavage. Amino acid se- quence from several peptides was obtained (see Fig. 4). The NH2-terminus was found to be blocked. The purity of the sequenced p150 was indicated by the fact that all the peptide sequences obtained were found in the deduced amino acid sequence from the cloned gene (see below). Isolation of the Gene Encoding p150 The internal peptide sequence obtained from p150 allowed us to use PCR to synthesize a corresponding segment of the gene encoding p150. From the sequence of the PCR product a specific anti-sense oligonucleotide was designed and used Downloaded from to screen a yeast genomic library. Five overlapping genomic clones were isolated. Shown in Fig. 4 is a 5,482-bp contigu- ous sequence obtained by bidirectional sequencing of the isolated clones. An ATG at position 743, which is flanked by conserved bases found at translational start sites (Hamilton et al., 1987), initiates an open reading frame 4,011 bp in www.jcb.org length. Flanking this region on the 5' and 3' sides are con- sensus sequences for transcription initiation (TATA) and ter- Figure3. Preparative chromatographic separation of proteins of en- mination (Struhl, 1987; Zaret and Sherman, 1982), respec- riched NPCs. To obtain fractions ofpl50 for protein sequence anal- tively. Southern analysis of yeast placed this ysis, enriched yeast NPCs were solubilized in SDS and polypep- on April 12, 2006 gene on 13 (data not shown). tides separated by sequential SDS-hydroxylapatite (SDS-HA) (A) and C-4 HPLC (B) chromatography. Polypeptides eluted from the Analysis of the Deduced Amino Acid Sequence of plSO SDS-HA column were separated by SDS-PAGE (every other frac- tion starting with number 22) and visualized by Coomassie blue The open reading frame of the gene encoding p150 is 1,337 stain (A). The position of p150 is indicated by an arrowhead. SDS- amino acid residues long with a calculated molecular mass HA fractions containing the majority of p150 (Fractions 33-39) of 151,670 Daltons, in agreement with the mass of p150 esti- were pooled and the polypeptides further separated by C-4 HPLC mated by SDS-PAGE. 13 internal peptide sequences ob- chromatography. Analysis of the column eluate (every other frac- tained from p150 are represented in the deduced amino acid tion beginning with number 35) by SDS-PAGEand Coomassie blue sequence of this gene, further establishing its identity (Fig. staining is shown in B. The position of p150 is again indicated by 4). On the basis of its apparent localization to the pore mem- an arrowhead. Molecular mass markers in kilodaltons are shown brane domain and its DNA-deduced molecular mass (~152 to the right of each gel. kD) we propose to term p150 as POM152. The integral membrane character of POM152 suggested these sites is glycosylated. This is further supported by the that it contains one or more transmembrane segments. Anal- observation that endoglycosidase H digestion of POM152 ysis of the hydrophobicity of the deduced amino acid se- abolished Con A binding and reduced its apparent molecular quence of POM152, conducted using the method of Kyte and mass by "~3 kD (data not shown). Fortuitously, peptide se- Doolittle (1982) (data not shown), identified a 19-amino quence was obtained from a fragment of POM152 containing acid segment (residues 175-195) (see Fig. 4) lacking charged a putative glycosylation site located at residue 280 (see Fig. residues and of sufficient hydrophobicity to function as a 4). The sequence of this fragment proceeds normally from transmembrane segment (Kyte and Doolittle, 1982). Consis- residue 270 to 302 but is blank at the position of the tent with this assignment, this segment is bordered by predicted asparagine (residue 280) (data not shown). This charged amino acid residues. suggests that in fact residue 280 is N-glycosylated (Evans et The deduced amino acid sequence of POM152 contains al., 1988). five consensus sites (NXS/T) for N-linked oligosaccharide The deduced amino acid sequence of POM152 appears addition, two on the NH~-terminal side of the transmem- unique by comparison with currently available protein data brane segment and three on the COOH-terminal side. The bases. No significant similarity exists between POM152 and binding of Con A to POM152 suggests that at least one of the two identified mammalian pore membrane proteins,

Wozniak et al. POM 152, a Yeast Pore Membrane Protein 35 GTCC~¢GqTGTT~CCGTA~GAGC~,ATGTTT TATTTCCAGATGATGCTAACATAkC~,CA~CTTC~T~T~T~CTTATAT~CTG 84 T TTTC~CCA¢GA~C,ACCAAATATCGAGGACTGCTTTGTCTATATCCATCCGACT TCAAT T TTAA~TAATTTGGG~T ~ACC~TA~T~A~A~T~T~T~C~AT~T~ TT 244 TAT&'TGACATCGCA~GCACCCCCTTAGCGAACATAG~GC,~CTTCTTTTGACATACAGTAKACC~CT~C~GT~ ~T~C~TT~TATCACCTAC~C G~A~G~A~T~T~CTA~T~CA 394 CAGTTGACAACGATCTCAAGATTGGTT ~TTTAA~TCCTATAGCAGTGCATCAGAAG~AG~U~G~T ~TT~TTAT~C~~C~T~~T~CG 544 CTGGTATTATAC.AAATAGAGTTTCATTATATAAC~CATATAGGAAGAATACATTATTCt'TATCT*~C~CATC~T~G~T~T~T~C~C~T~T~AT~ATAG~CTATGTAT~ATGT 594 CATTATATAC GTAGTCAATAACTTTATACGTTTTTTTTATTGAACA~TAAGAATTGCTTAA GACATAAT~C ~A~C~C~G~TTAT~ATCATACCA~T~TAT~C~GTATA 844

1 I0 20 30 40 50 M E H R Y N V F N D T P R G N H W M G S 8 V S G S P R P 8 Y S S R P N V N T T R R F Q ¥ $ D D £ P A ATGC~%~.ACAGATAT~GTGTTT~U%TC~TACTCC~C,ACC~CCATTGGATGGG CAGTTC TGTGT CAG~T ~C~CGTC ~TCTTATA~A~ CGTC ~TGT~CA~C~C~T~C~TATA~ ~CGA~CC~T 994

80 70 80 90 I00 E K I R P L R S R $ F K S T E S N I S D E K S R I S E R D S K D R Y I N G D K K V D I Y S L P L I S GAC~T CC GACCTTTACGCTCGAGGAG CT TTAAAAGTACTC.KAAGTAACATAAGCGATGA~TCAAGGATATCT G~ACGTGACA~GAC C~T~A~T ~T~GGTAGACA~TATT~CTGC C TC T~TATC A 1144 Pore

11o 12o 13o 14o 15o Side T [3 v L E I s K Q R T F A v I L F L I I Q C Y K I Y D L V I L K S G L P L S G L L F ~ N Y R F N F I AC~TGTTTTAC4%AATTTCCA~ACAAAGC,AC ATTTGCGGTC~%TAT T gfTTTTAATAAT TCAATG'~T&TA~TATAT GACCT~ T~TAC ~TCGG ~TTAC CG C~T CGGGT~AC TGT~CTATC~T TT~ T~TA 1294 +

160 ]70 180 180 200 S X Y F I I D 8 F F L Y V L P S F N I P R L T F K[~ W V V Y ~ ~ 1 L k M L L L N I F I 8 S[ D H E F V Tl~ TCC~,~GTATTTC.J~TATAC4%TTCC T~ TTCCT~A~TTTTAC CATC~TT~CATTCCTAOGTTC~CT T~C~T~G~G~TATC~TTAC~A~CTACTAT~A~ATAT~ATCACGA~T CG TT 1444 .i 210 220 230 240 250 L I S L I M T T W R K L Y T K E L $ V T G S A I N H H R I F D ~ ~ A ~ F M G A L T I K I L P E N T A Cis~ TT C.ATTTCATTAATTATGACCACATGGAGA~CTTTATACGA~T TAAG TG TAACAGGTTCAG~TT~TCAT C~A~ ~TT~CCG~CATT~G~C T~CTAC ~ ~C~CT 1594 Side

260 270 28-0 290 30O M F N P L H E S ¥ C ;~ P M D T N L F K I N S I D V P I R 1 N S T E E I £ Y I E L £ Y RVD L Y T N S V AT &'TTT~AT~CT~T'~CATC4~T C.%TATTG~n'T~CCAT(~C,~TACC..%ATCTI~fTT~T T~A~C~%T'~CGTACCCA~A~T~CT ~ ~TATA~ ~T ~TATA~TC~TACAC~ C~TA 1744

310 320 330 340 350 E L R S L S K K ~ F K I I D N P K S F L K K 0 Q $ V L K S H S N D ~ ~ E G S T I R Y L A V T L Q D I GAGTTACGGTCTTTGAGTAAC~GACT TCAAAATC.~TCGATAACCCCAAATCCTTTTTAA~T CAGTCAGTTCT~GT~CATT ~CGAT T~G~A~ C~TATT~GT~CT~ATATT 1894

3~0 870 3~0 390 400 G ~ M Q I K K I V D $ K K L ~ L K I H ~ S ~{ L V V P Y C P I A $ I T G T G S N D ~ C I G D S D N V S C-GCTTTTATC~TC~TTGTTGATTCAAA~TGAATTTAAKC~TCCATCAGTCACACTT~GTTG TAC CCTAtT GTC~TT G~T~A~A~G ~G~T~TA~T~A~G~ ~TT~T~T~CT 2044

410 420 430 440 450 F E I O G V P P M K L A Y $ K I V N G ~ T F S Y V D $ S L ~ P E Y F E S P L Q S S K S K Q $ F T Q G TTTC~%~%~GTGCCCCC.AATC, A~J%TTA(~CATACT CTAAC,AT~TTAATC, G'i~AAACTTTTTCATATGTAG~T~A~C~C~TATT~ GAGTCTC ~TTGC~ T ~T~C~ TT TACTC~GA 2194

460 470 480 490 500 £ L N D L K W G R N O P V N I N L D S $ I T 0 D G K F A Y K I D K I T D G L G N V V D F T S L P E E GA~TAAACGATTTC~TGGGGAAGAAACCAAC CTGT TAATATTAATTTAGACTCTTCCATTACCCAGGAC GGCAAATTTGCATAT~GAT~T~ GAT G~CTGG~TG~GTG~T T~A~T~CT~C ~G~ 2344

510 820 530 540 550 L K K R Y D L $ Y N F N V H E V 8 R A A L E E R F D P K S P T K ~ S I A 3 v F E E I K N W I S 0 I P TTAAA~GCTATGATCTATCTTATAATTTCA~TGTTCATC~CA~GTGCAGCC~TAGA~GATTTGAT C~ T~C~A~T ~A~ G~A~ G~T~TC~CT ~TC~T~CA 2494

860 870 580 590 800 M V I S L S ¥ T D A ~ D K S K K I M R V T T 0 S L T K V L Q A D L P G S Y N L E Y I E S K ? C P G E TAT~ATTAC~TATCCTATACTGATC~CAAGTCC.AA~CATAATC~%ATGT T~CTACAGATTCT TT~CC~O ~T TCC~GCGCATC~CCG~T~TAT~CTTA~TATA~T~T ~TGTCC~TG~ 2644 Downloaded from 810 820 630 640 650 I V G K S N V L V T M P V A P T M E V K S F P I L D Q C V G Q v 0 L N F E L S F T G A P P Y Y Y N T ATTGTGGGT~%ATCAAACGTTCTTG TAACTATGCCAGTTGCACCTACTATGGAAGT TAAATCATTTCCAATATTGGACCAGTGTGT~ ~GG ~ G~C T~CT ~ G~C TATCT TTTA~ G~G ~C ~C~TACTA~AT~CT 2794

680 670 880 690 700 K I Y ~ L E N G £ ~ K L Y D A K R ¥ T S E G T R N R F S Y $ P P K E G N Y E I V F D T V S N K L F T A~%TC~AT~%G~TC~CG~C~A%AC~A~AGTTATATGATGC~C.AC~ATA~-%CCTCTG~-%GGTAC~T~T~ACC TATA~C~C~G~ TAT~G~T~GATA~T~T~TTATT~CT 2944

710 720 730 740 750 E P I K L ~ P V K E Y T F K T S M R V K P 8 A S L K L H H D L K L C L G D H S S V P V A L K G Q G P G~CAATCA~ATT~CCTGTAA~TATACTTTCAACACATCAATGAGGG TGA~C CAAGCGCATCAC TA~T TACACCAT GATTTGAAAC~TGTT ~GGT~C CACA~AGTGTC C ~GTAG~C T~G~ C T 3094

780 770 780 790 800 F T L T Y D I I E T F S S K R K T F E I K £ I K T N E ¥ V I K T P V F T T G G D Y I L $ L V S I K D www.jcb.org TTTACGTTAACATATGATATCATT GAKACTTT TTCTAGCAAGAGG~CTTTTC~TTAAAGA~TAAAAACC]tACGAATATG TCATT~C ~G ~T~ ACTA~ G~G~ GATTATA~CTKT~ TTGG ~ TCTAT~AT 3244

~IO 820 830 ~40 850 $ T 0 C V V 0 L $ 0 P D k K I 0 V R R D I P s A k ~ N ~ F ~ P ~ K ~ k x I K H C S V T E I ~ L K L $ TCTACCGGCTGTGTAGTTGGACTCAGCCAACCAC~TC~TA~TACAGGTGAGGAGAGATATTCC.%TCTGCTG~T~TT~T~C~ATC~G~G~GCACGGTT ~ ~CACT~TT ~CGC T~TT~GT 3394

880 870 880 890 900 G E G P F T V K ~ K H M D Y 0 G N I V K E F E N K F Q N S Y K P A L K V S K 8 0 L Y Q L V D I R D S on April 12, 2006 G GAC,AGGGGCCATT~ACCGTTAAGTTTA~AC2%TGGATTACCATGC,~CATTGT ~.%TTTC~C~C*~TA~TAT~C~T TG~GT~GT~G~C~ TACC ~T TGG~TATTCG TGA~ CA 3544

910 920 930 940 950 S 0 Q G N V I Y R N S L Y K V S F L E K P ~ F A I Q D N H H I T K V T E N L F S K E E V C ~ G M E G A STTGCC~%GGTRATGTC, ATT TACC GC,J~%C.%GTCTGTAT~GGTATCTTT CTTG~4%C4~GCC~%~ATTTGC~C~C~T ~TC~ATTA~CT~CGG~TT TATTCT~G~GTCT ~ C~G~T~GGT 3694

980 970 980 990 i000 T V D L A L F G S P P F I L E Y D L M A P N G H I $ r K K I Q V A T ~ Y A S L K L P N Q I P G E Y I AC~GTTC~TTTG~CTATTTC.GTTCTCC~CAT TCATATTAGAATATGATT TGATGGCA¢CCAACGGTC ATATTT~A~ ~GG ~G~TACGCTT ~CTG~C~C ~TC~ C~G ~A~T A 3844

IO10 1020 1030 1040 1050 T T I K A I ? D G M ¥ G E $ D I H F R E H ~ S E L I I K ~ T V K P I P D V A F A D G G K T L R A C A ACTACT~TTAAGC.CCATCT TCGATG~TTATGGTO~GC~TACATTT TAC~ATCAGT~G~C ~AT~CAGA~GT~C~A~C~C GTC G~ G~G~TG~G~T TGC~TGCATGTGCT 3994

1080 1070 1080 1090 II00 A N V D Q I 8 r L E P l N L K F L 0 0 E S P F 8 1 T r S V Y H ~ S T 8 R T 0 Q Y T l D N Z D S E N F GCT#~%TGTAC4%TC~TC TCATTTTTC~G~ACC~AT~TTTGA~g~TT TTTAC2~GCC,~C, CCCAT TT TCAATCA~T ~G~TATC~G~ACCA~A~A~GATCAGTATACCA~ GAC~ATTGACTCAGA~TT 4144

Iii0 1120 1130 1140 1150 S F E K L ¥ E G M K L G N H A I T I D S V V D A N G C V N S L I S G P R N O I L V $ I T D A P ~ I H TCATTTC,ARARGTTATATGMtGGGRTC, ARGTTAGGTAACCATGCCATTACTATTCOt TTC TGTTGT TGRCGCAAAT ~TT or GTT~A~C~ATAT~ G~C CGC~T C~ C~ GTGT~TTA~ GATG~CC~TACAT 4284

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1210 1220 1230 1240 1280 P G I 1 S I T s L Q D S $ S Q 0 I V D F T N P M L K S E F 0 0 L 8 L N [ H P I P $ V T V S ~ G N T V CC~.~CATTATATCTAT~CTTCTTTACAGGATTCATCATCACAATGCAT TGTCGACTTTACGAAC~CTAAATTAAAGAGTGA~T~TG~ TTAT~TT~ATACATC~A ~CC TTCCGT~G~ TCTC~G~ACGT T 4594

1260 I~70 1280 1290 1300 T Z 0 1 R £ 0 D ~ k E '4 1 F S F E 0 T P 8 F 8 L T Y V R T E £ T D u K ~ G I( R R s 0 V v ~ T 8 K v T ACTGA~ATAT~GCC~GGTGACCAGGC T~TAATCTTTTCGTTTGA~GTAC CCCACC&TTCTCAC T~ACTT&T GT&~GGACAGAA~TG~CACG~T CGC~G~ACGCAT~T ~CT 4744

1310 1320 1330 1337 D I M S M E Y K V I T $ L 0 G T M £ A ] E I T 0 A Y C F A K N D L F F N N . GATATATATT~TCATGAATACA~TAATTACAAGCTTG~GT&CCTAC~GATTGAKATT&CAGATGCTTATTGTTTT C~CA~T~T~T ~ T~T~T~TATTAC~O~C~TT ~T~TTCCC 4884

GCAATATTTTTGTACAATTGTAATGTATAATATATCTCTGTACATCAGTATTAAAATACTTA~GCGA~OACGCCT ~A~G~T~T~T~ ~T~T ~TTAT~C~ TC~C~TTAT~T C~T~T~TA 5044 ACTCGAAA~AGTGTAGGTGAATGAATAGA~GAGCGC TGAGACCTTTGTCCTTTACC~TCTAT~T~G ~C~A~C~A~T~TG~TATCO~TAC~AT~T~GG 5184 TC'I'C,AAGTTCATAGGGTG~C,lttAGG¢ OACTAJ~TTATTACTTT OGACGCATAT&ATACACTATACGCCA~CTTOCCGTCATG~C ~T~T~A~G ~C ~TAT ~A~G~TC~TCGA~T T~C~C~T 5344 TTTC CACACGTTTTTAAA~CTC~GARGACTATCCCCAATACGG~%ATATTC TGGTATC~G~CCGAACAATGGTGGT CCATCT TGATT~CG ~T~G~C~C ~C~GATG~ATGAT~T 5483

Figure 4. Nucleotide and deduced amino acid sequence of the POM152 locus. Nucleotides are numbered on the right. Amino acid residues, represented by their one letter code, are numbered above their symbol at intervals of 10 beginning with the initiator Met and extending through residue 1,337 of the open reading frame. Sequences obtained by peptide sequencing of POM152 are underlined. The potential transmembrane segment is boxed. The asparagine (residue 280) believed to be modified by oligosaccharide addition is indicated by a star. The point of insertion of the hemagglutinin epitope tag following amino acid residue 293 is indicated by an arrowhead. The predicted topology of POM152 in the pore membrane is shown on the right.

The Journal of Cell Biology, Volume 125, 1994 36 A was replaced with a DNA fragment encoding the HIS3 gene (Fig. 6 A). Stable His + transformants were selected and POM152 ~-~FLY~L~-~-~C-'~F~-'~ 176 analyzed by Southern blotting to identify those with the cor- POM121 ITFISNTMT)PTFNIPIFS(S_.A_~AII(pI 924 rect replacement. One transformant was sporulated and tetrads were dissected (Fig. 6 B). In all cases the segregants B were viable and grew equally well. For all segregants, I-Iis+::His- segregated 2:2. Southern blotting of one set of 413 segregants confirmed the disruption (Fig. 6 C). A compari- G V LIN F('--'iL S FIT g AlP P Y Y YIN T 650 son of both haploid cells carrying the disruption and wild- -HS(SVP. VALIKGO~PFTLIT~] 755 type cells revealed no obvious morphological differences IKI, K~.J~J- v TIE I P L K L,S G C~le F Z VIKIFI 859 when examined by light and electron microscopy (data not shown). Both mutant and wild-type cells also grew equally -PX ~r-UOCEISeFSIITIF I 1077 well at temperatures ranging from 17 to 37°C (data not y -- YEI~vV],,0 L,N e VIA f' F, ZI*IVl 1,78 shown). L~ I R EGL~_DJ- 0 A 1F S F!E G TIP PF S LIT~ 1276 lmmunolocalization of Epitope-tagged POMI52 C-G ..... V---L-G--PF---Y :o.=o,s~, To establish the cellular distribution of POM152, the local- Figure 5. Alignmentsof POM152 with POM121 and of the POM152 ization of epitope-tagged POM152 (POM152-HA) was ex- repeats. (A) A 19-amino acid residue segment of POM152 is com- amined by indirect immunofluorescence microscopy. The pared with a similar region of POM121 by the method of Pearson tag, which codes for two tandemly repeated, 10-amino acid and Lipman (1988). Identical or similar amino acids are boxed. The epitopes derived from the hernagglutinin antigen (HA), was position in the protein of the last residue in each segment is shown inserted at a unique BgllI site (see Fig. 4). Haploid cells with on the right. A single gap is used to improve the alignment. (B) a chromosomal disruption of POM152 (PMY17) were trans- Alignment (by the Pearson/Lipman method) of eight repeated seg- formed with a single copy plasmid (pPM1-HA) carrying the ments of ,,024 residues within POM152. Boxed are identical or similar residues present in five or more of the repeats. Gaps are in- epitope-tagged POM152 gene (pom152-2::HA) and its en- Downloaded from serted to improve the alignment. A consensus sequence is shown dogenous promoter to produce the strain PMY17-HA. Using below which is derived from identical residues in five or more an mAb against the tag (anti-HA), POM152-HA was visible repeats (shown in bold). The position of the last residue in each re- as patches along the surface of the nucleus (Fig. 7 A). This peat is shown at the right. pattern is characteristic for NPC proteins (Davis and Fink, 1990; Wente et al., 1992; Wimmer et ai., 1992).

Definitive evidence for the location of POM152 at the NPC www.jcb.org was obtained by immunoelectron microscopy. Spheroplasts gp210 and POM121, with the exception of a short region ad- from PMY17-HA cells were fixed and either embedded in jacent to the NH2-terminal side of the POM152 transmem- Lowicryl and sectioned or directly frozen and cryosectioned. brane segment that shows 43% similarity to amino acids Sections were probed with the mAb against the epitope tag on April 12, 2006 905-924 of POM121 (Fig. 5 A). When this segment of POM- and binding was detected with 10-nm colloidal gold particles 121 or POM152 was compared to the protein data bases, in- bound to goat anti-mouse IgG. The gold particles are local- cluding gp210 (Wozniak et ai., 1989) and the molecularly ized along the NE at the NPC (Fig. 8 A-C). The positions cloned (Davis and Fink, 1990; Nehrbass et ai., of over 200 gold particles were quantified on these sections 1990; Starr et al., 1990; Wente et al., 1992; W'tmmer et al., as described in Wente et al. (1992). This revealed a high de- 1992; Radu et al., 1993; Sukegawa and Blobel, 1993), no gree of label specificity for the NE and the NPC (Table I]). similar sequence was identified. The functional significance, In an attempt to sublocalize the native POM152, the en- if any, of this homology remains to be investigated. riched NPC fraction was probed with colloidal gold-labeled Finally, using dot matrix analyses we have identified eight Con A and visualized by negative stain electron microscopy repetitive segments in POM152, each ,024 amino acids in (Fig. 8 D). As POM152 is the predominant Con A-binding length. All eight segments lie on the COOH-terminal side of protein in the NPC fraction, the gold distribution should the transmembrane segment. The sequence of these repeat- reflect the localization of this protein. We observe that ing units and their alignments are shown in Fig. 5. With the ,050% of the gold particles (n = 661) were found attached exception of the distance between the first and second re- to the NPC rims (Fig. 8 D), representing a 50-fold higher peats, each of these repeating motifs begins at ,0100--amino specific activity (gold particles/rim2) over background. Both acid intervals. The regions between these repeats show no the specific labeling and the background were dramatically apparent similarity to one another. reduced by the presence of the competitive inhibitor methyl o~-o-mannopyranoside (data not shown). Thus, despite the POMI52 Deletion Mutants Are Viable polyspecific nature of the probe, it is likely that the binding Southern blotting of yeast genomic DNA cleaved with vari- of Con A to the NPC rim reflects the position of POM152, ous restriction enzymes showed that the POM152 gene is consistent with it being a pore membrane protein. present in a single copy per haploid genome (data not shown). To define the phenotype associated with the disrup- Overexpression of POM152 Inhibits Cell Growth tion of this gene, we deleted it in the diploid strain W303 by gene replacement. This was accomplished by integrative We have investigated the effects of POM152 overproduc- transformation using a DNA fragment in which a 3.6-kb tion on the growth of diploid W303 cells, pom152::HA was MboI/SalI fragment from within the POM152 reading frame placed under the control of the inducible GALl promoter in

Wozniak et al. POM 152, a Yeast Pore Membrane Protein 37 Figure 6. Deletion and disrup- tion of the POMI52 gene. The POM152 gene was disrupted by deleting an MboI/SalI frag- A ment within the open reading frame (represented by the BamXl Mbol BW II ,.~11 BamHI thick black line) and replacing it with the gene for the HIS3 selectable marker (A). Inte- grative transformation of the diploid yeast strain W303 was performed with a BamHI frag- ment from this construct. A B~Hi HI83 Xhot heterozygous diploid strain was then sporulated and the tetrads were dissected. B shows the haploid segregants of six tetrads. In each case all four spores were viable. To confirm the success of the dis- ruption Southern blots were performed using BamHI- cut genomic DNA from four hap- loid segregants (C, lanes 2-5) and the parent diploid strain (C, lane 1). The transformed diploid strain contains both the wild-type gene (6.6 kb) Downloaded from and the disrupted gene (4.4 kb). Both His+ (i.e., dis- rupted) haploids (C, lanes 4 and 5) lack the wild-type POMI52 gene. www.jcb.org on April 12, 2006

pom152-2::HA Figure 7. Localization of epitope-tagged POM152 by indirect immunofluorescence microscopy. Spheroplasts from the haploid strain PMY17-HA (containing pom152-2::HA) expressing the HA-tagged POM152 gene product were fixed, permeabilized, and probed with a mAb (12CA5) directed against the HA epitope. Binding to POM152-HA was visualized with FITC-labeled goat anti-mouse IgG (Anti-HA). DNA-binding DAPI stain was used to define the nucleus in the same cells (DAPI). POM152-HA is visible along the nuclear surface in a punctate pattern characteristic of NPC localization. Bar, 5/~m.

The Journal of Cell Biology, Volume 125, 1994 38 Downloaded from www.jcb.org on April 12, 2006

Figure 8. Localization of epitope-tagged POM152 to the NPC by immunoelectron microscopy. Spheroplasts from the haploid strain PMY17- HA expressing the HA-tagged POM152 gene product were fixed and either embedded in Lowicryl (A) or prepared for cryosectioning (B and C). Sections were probed with mAb 12CA5 and binding was dewzted with goat anti-mouse antibodies bound to 10-nm gold particles. Gold particles are visible along the nuclear envelope in association with the NPCs. Presented in D is an electron micrograph of a negatively stained enriched NPC fraction which had been probed with Con A linked to 20-nm colloidal gold particles. The particles are visible around the rim of the isolated NPCs. Bars, 0.2 #m.

Wozniak et al. POM 152, a Yeast Pore Membrane Protein 39 Table II. Distribution of Gold Particles on Immuno-stained work (Fig. 9 B). No signal was observed with the anti-HA Spheroplasts Expressing POM152 : :HA antibody in cells containing the pRS426 plasmid (Fig. 9 C). Location Total number Density Expressed POM152 Localizes to Mammalian Nucleoplasm 16 0.10 Pore Membranes NPCs 114 1.46 Cytoplasm 77 0.08 We have examined the subcellular distribution of the pom- 152::HA gene product in mouse 3T3 cells to determine The number and location of lO-nm gold particles were determined for 100 whether it would accurately localize to the nuclear pore spheroplast thin sections. The densityof the gold particleswas calculatedas described (Wente et al., 1992). membrane. To do this the complete open reading frame of pom152::HA was inserted into a transient eukaryotic expres- sion vector (pSVL). Plasmid DNA was introduced into 3T3 cells by direct microinjection into the nuclei of subconfluent, unsynchronized cultures. The localization of the expressed pom152::HA gene product was evaluated by immunofluores- the high-copy, 2-#m plasmid pRS426. The resulting plasmid cence using the mAb against the tag (Fig. 10, Anti-HA) 21 h (termed pPMGai) and the pRS426 vector alone were in- after injection. Indeed, POM152:HA was visible in a char- dependently transformed into W303 cells and the growth of acteristic punctate pore membrane pattern (Wozniak and individual transformants examined on medium containing Blobel, 1992) both when viewing the nuclear surface (Fig. either glucose or galactose (Fig. 9 A). In cells containing the 10 A) and the nuclear rim (Fig. 10 B). No staining was visi- pPMGal plasmid, gaiactose-induced overexpression of the ble in cells not expressing the pomJ52::HA gene product pom152::HA gene product markedly inhibited cell growth (data not shown). These data suggest that yeast POM152 can (Fig. 9 A; compare the growth of three strains containing the be accurately sorted and retained within the pore membrane pPMGal plasmid on SM-URA galactose to their growth on domain of mammalian cells. At high levels ofpoml52::HA SM-URA glucose). When one of the overexpressing strains expression an additional endoplasmic reticulum-like stain-

(PMGall) (Fig. 9 B) was examined by indirect immunofluo- ing pattern was also observed (data not shown). This is simi- Downloaded from rescence using the anti-HA antibody, POM152-HA was visi- lar to what has been previously observed with elevated ex- ble along the periphery of the nucleus and in patches adja- pression levels of the mammalian pore membrane protein cent to the plasma membrane suggesting that the protein is gp210 (Wozniak and Blobel, 1992, unpublished data) and is largely present in the NE and the endoplasmic reticulum net- consistent with the movement of POM152 to the pore mem- www.jcb.org

Figure 9. Overexpression of

POM152. Diploid yeast W303 on April 12, 2006 cells were transformed with the plasmid pRS426 or pPM- Gal (pRS426 containing pom- 152::HA under the control of the Gall promoter). Cells de- rived from individual trans- formants were streaked onto SM-URA galactose and SM- URA glucose plates and tested for their ability to grow (A). As shown in A, cells contain- ing pRS426 (v) grew equally well on both Galactose- and Giucose-cont~ufmg medium. However,the growth of strains containing the pPMGai plas- mid (1, 2, and 3) was markedly inhibited on gaiactose plates with no colony formation visi- ble. The overexpression of the pom152::HA gene product was confirmed by indirect im- munofluorescence. Cells from a strain carrying the pPMGal plasmid (B) or a control strain with the pRS426 plasmid (C) were grown for l0 h in SM-URA galactose, processed for immunofluorescence, and probed with the anti-HA antibody (12CA5). Binding was visualized with FITC-labeled anti-mouse IgG (Anti-HA). DAPI stain was used to define the nucleus in the same cells (DAP/). In the PMGall strain, POM152-HA was visible in the nuclear envelope and adjacent to the plasma membrane in an endoplasmic reticulum-type pattern (B, Anti-HA). No staining was visible in the pRS426-containing strain (C, Anti-HA). Bar, 5 #m.

The Journal of Cell Biology,Volume 125, 1994 40 Figure 10. POM152-HA is targeted to the pore membrane domain of mouse 31"3 cells. 3"1"3cells expressing the pom152::HA gene product were fixed, permeabilized, and probed with rnAb 12CA5. Binding to POM152-HA was visualized with Texas red-labeled goat anti-mouse IgG (Ant/-HA). POM152-HA is visible at densely packed points along the nuclear surface when the focal plane is tangential to the nucleus (Nuclear surface) or as a punctate ring when the focal plane passes through the center of the nucleus (Nuclear rim). This pattern is character- istic of nuclear pore proteins. Bar, 10 ttm.

brane domain following its integration into the endoplasmic ture (by 180 °) of the pore membrane on its cisternal side. Downloaded from reticulum membrane. Similarly, the cisternally interacting domains of POM152 or its homolog(s) (see below) may contribute, at least in part, Discussion either to the lumenal spoke domains or radial arms (see introduction). To date, the pore membrane domain of the yeast NE remains The NH2-terminai portion (residues 1-175) of POM152 largely undefined. We report here the identification and the that precedes its transmembrane segment is likely to be ex- www.jcb.org characterization of an integral membrane protein of this do- posed on the pore side of the pore membrane. A small seg- main and term it POM152. POM152 coenriched with yeast ment of the region (residues 158-176) shows a 43 % similar- NPCs indicating that it interacts with the NPC proteins ity (see Fig. 6 A) with a topologically equivalent (i.e., pore

(termed nucleoporins or NUPs), directly or indirectly. That side exposed) region of the rat POM121 (residues 905-924). on April 12, 2006 this protein was originally observed as a coenriching constit- The functional significance of this homology, if any, remains uent of the highly enriched NPC fraction (Rout and Blobel, to be established. 1993) further strengthens the likelihood that many of the POM152 is not synthesized with a cleavable signal se- other 80-90 coenriching proteins in this fraction are pore quence for integration into the endoplasmic reticulum. As in membrane (POMs) and nuclear pore complex (NUPs) com- the case of other type II membrane proteins, the transmem- ponents. brane segment of POM152 is likely to function both as a sig- Although we have not experimentally determined the to- nal sequence and as a stop transfer sequence to integrate the pology of POM152, our data suggest that it contains a single protein into the bilayer of the endoplasmic reticulum. It re- transmembrane segment. The COOH-terminai region (resi- mains to be determined which domain of POM152 serves as dues 196-1,337) of POM152 is likely to be entirely posi- the sorting determinant to localize it to the pore membrane tioned on the cisternal side of the pore membrane; this region domain. For gp210, the dominant sorting determinant has re- contains three consensus sites for N-linked glycosylation, cently been localized to its transmembrane segment (Woz- and at least one of these sites appears to be glycosylated (see niak and Blobel, 1992). Our observation here that POM152, results), explaining its reactivity with Con A. Although this when expressed in mammalian cells, is correctly sorted to cisternal side-exposed region of POM152 shows no sequence the pore membrane domain of these cells suggests that this similarity to other proteins in the data base, it shows eight pathway is conserved between yeast and mammals. More- repetitive segments of • 24 residues (see Fig. 6 B). Of note over, the conserved sorting suggests that POM152 might is a regularly spaced cysteine in five of the eight repeats. substitute for a mammalian structural and/or functional Moreover, all but one of these repeats begins at intervals homolog. ,,o100 residues apart. These repetitive segments might be in- Surprisingly, a yeast strain in which the POM152 gene was volved in homophilic or heterophilic interactions on the cis- deleted was viable and its growth indistinguishable from that ternal side of the pore membrane. Homophilic or heterophilic of wild-type ceils at a wide range of temperatures. It is possi- interactions via repetitive (and topologically equivalent) do- ble, however, that other growth conditions would reveal a mains are characteristic of many cell surface adhesion mole- phenotype. This remains to be investigated. Alternatively, a cules (Edelman and Crossin, 1991). In the case of POM152, functionally homologous protein(s) may be present within side interactions may help stabilize the sharply bent struc- the nuclear pore which can compensate for the loss of

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