POM152 Is an Integral Protein of the Pore Membrane Domain of the Yeast Nuclear Envelope

POM152 Is an Integral Protein of the Pore Membrane Domain of the Yeast Nuclear Envelope 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 nuclear pore 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 proteins (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 isolation 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 gene 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.

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