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3449 Journal of Cell Science 107, 3449-3459 (1994) Printed in Great Britain © The Company of Biologists Limited 1994 Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles Christopher R. Cowles, Scott D. Emr* and Bruce F. Horazdovsky† Division of Cellular and Molecular Medicine & Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668, USA *Author for correspondence †Present address: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038, USA SUMMARY Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete vacuolar hydrolases. A small subset of vps mutants exhibit a temperature-conditional growth phenotype and show a severe defect in the localization of soluble vacuolar proteins, yet maintain a near-normal vacuole structure. Here, we report on the cloning and characterization of the gene affected in one of these mutants, VPS45, which has been found to encode a member of a protein family that includes the yeast proteins Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and Rop from other eukaryotic organisms. These proteins are thought to participate in vesicle-mediated protein transport events. Polyclonal antiserum raised against a TrpE-Vps45 fusion protein specifically detects a stable 67 kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that the majority of Vps45p is associated with a high-speed membrane pellet fraction that includes Golgi, transport vesicles and, potentially, endosomal membranes. Significantly, this fraction lacks ER, vacuole and plasma membranes. Overexpression of Vps45p saturates the sites with which Vps45p associates. A vps45 null mutant accumulates vesicles, many of which were found to be present in large clusters. This accumulation of potential transport vesicles indicates that Vps45p may facilitate the targeting and/or fusion of these vesicles in the vacuolar protein sorting pathway. INTRODUCTION fragmented, while class C mutants lack any identifiable vacuole structures. Class D vps mutants are morphologically similar to class A strains but have a large, single vacuolar structure and exhibit defects in mother-to-daughter cell vacuolar inheritance. Class E mutants contain a novel endosome-like compartment in addition to normal vacuoles, and class F mutants contain a single large vacuole structure that is encircled by smaller, fragmented vacuoles. Even though some vps mutants exhibit severe defects in vacuolar morphology, the vast majority of vps mutants (>75%) contain a normal or near-normal vacuole structure (classes A, D, E and F). This suggests that these mutants are competent to construct and maintain a vacuole structure, yet have specific protein targeting and sorting defects. Some of these defects may involve the disruption of specific vesicular trafficking events that are responsible for the delivery of distinct subsets of vacuolar proteins from a late Golgi compartment to the vacuole. Analysis of protein movement through various stages of both the secretory and vacuolar protein localization pathways has uncovered a number of protein families that direct common events (i.e., transport vesicle targeting and fusion) at each stage of the transport process. One such family includes the yeast rab proteins Ypt1p, Vps21p and Sec4p. These small GTP-binding proteins appear to be involved in the targeting and/or fusion of vesicular transport intermedi- The localization of proteins to the lysosome-like vacuole of Saccharomyces cerevisiae is a complex process and involves several distinct targeting and delivery events. Like secreted proteins, vacuolar proteins are first translocated into the endoplasmic reticulum (ER), and subsequently transit to and through the compartments of the Golgi complex (Stevens et al., 1982). In a late Golgi compartment, vacuolar proteins are then actively sorted away from the secretory protein flow (Graham and Emr, 1991) and delivered to the vacuole via a prevacuolar endosome intermediate (Vida et al., 1993). In order to identify the cellular machinery involved in Golgi-tovacuole protein sorting and delivery events, several mutant selection schemes have been designed that detect mislocalization of soluble vacuolar proenzymes (Bankaitis et al., 1986; Rothman and Stevens, 1986; Robinson et al., 1988; Rothman et al., 1989). Using these selection schemes, a large number of mutants have been identified that missort and secrete vacuolar proteins. Together, these vacuolar protein sorting (vps) mutants define greater than 40 complementation groups that have been divided into six distinct morphological classes (Banta et al., 1988; Raymond et al., 1992). Class A vps mutants display wild-type morphology: one to three large vacuole structures are present in each cell. The vacuoles of class B vps mutants are Key words: VPS45, SEC1, vacuole, protein sorting, transport vesicle 3450 C. R. Cowles, S. D. Emr and B. F. Horazdovsky ates at distinct stages of the transport pathway. Ypt1p is involved in ER-to-Golgi vesicle movement (Goud et al., 1988). Vps21p functions in the vacuolar protein sorting pathway (Golgi to vacuole; Horazdovsky et al., 1994), and Sec4p is required for delivery of secretory vesicles from the Golgi to the plasma membrane (Bruno et al., 1988). Another group of proteins, the Sec1 family, also appears to function in vesicle targeting or fusion. In yeast, the Sly1, Sec1 and Vps33 (Slp1) proteins are required for protein delivery from ER to Golgi, Golgi to plasma membrane, and Golgi to vacuole, respectively (Novick et al., 1980; Banta et al., 1990; Wada et al., 1990; Dascher et al., 1991). Distinct Sec1p family members also have been implicated to function in the vesicular trafficking events of other eukaryotes. In mammalian neuronal cells, n-Sec1 has been shown to interact with the plasma membrane protein syntaxin (Hata et al., 1993; Garcia et al., 1994; Pevsner et al., 1994). Syntaxin is thought to serve as one of the receptor molecules involved in the docking of synaptic vesicles with the plasmalemma (Söllner et al., 1993a,b). UNC18 has been proposed to serve a similar function in Caenorhabditis elegans (Hosono et al., 1992; Pelham, 1993). Though members of the Sec1 protein family and the rab-like GTP-binding proteins clearly play critical roles in vesicle-mediated protein trafficking, their exact functions remain unclear. Here we report the characterization of one of the VPS gene products involved in delivery of proteins to the yeast vacuole. VPS45 encodes a 67 kDa homolog of Sec1p. Subcellular localization of Vps45p suggests that it is peripherally associated with cellular membranes (potentially including Golgi and endosomal membranes, as well as membrane vesicles). vps45 mutants missort multiple vacuolar hydrolases, are temperature sensitive for growth, and exhibit a class D vacuole morphology. Interestingly, vps45 deletion mutants accumulate what appear to represent aggregates of intermediate transport vesicles. No other characterized vps mutants have been shown to exhibit this phenotype. Our findings thus suggest a unique role for Vps45p in vesicle targeting and/or fusion in the vacuolar protein sorting pathway. MATERIALS AND METHODS Materials LB and M9 media supplemented with antibiotics and amino acids as required (Miller, 1972) were used for propagation of Escherichia coli. Yeast extract-peptone-dextrose (YPD), yeast extract-peptone-fructose (YPF) or synthetic dextrose (SD) was employed for growth of S. cerevisiae and supplemented as needed (Sherman et al., 1979). Boehringer Mannheim, New England Biolabs, or Stratagene restriction and modifying enzymes were used in this study. Sequenase Version 2.0 was obtained from US Biochemical Corp. Zymolyase 100-T (Kirin Brewery Co.) was purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Glusulase was received from the DuPont Co. 5-Bromo-4chloro-3-indoyl-β-D-galactoside, phenylmethylsulfonyl fluoride, α2macroglobulin, aprotinin, leupeptin, pepstatin and isopropyl-β-Dthiogalactopyranoside were from Boehringer Mannheim. Tran 35S-label was obtained from ICN Biochemicals, while [α-35S]dATP was purchased from Amersham Corp. Production of antisera to CPY, ALP and PrA has been described previously (Klionsky et al., 1988; Klionsky and Emr, 1989). All other reagents were purchased from Sigma. Plasmid constructions Recombinant DNA manipulations employed in the construction of plasmids were performed as described previously (Maniatis et al., 1989), with the exception of DNA fragment isolations carried out by the glass bead method of Vogelstein and Gillespie (1979). The CENbased plasmid pVPS45-10 was generated by inserting the SmaI-PvuII fragment of library plasmid pVPS45-1 (containing VPS45, refer to Fig. 1A) into the SmaI site of pRS414 (Sikorski and Heiter, 1989). The same SmaI-PvuII fragment was also inserted into the SmaI site of pRS424 (Sikorski and Heiter, 1989) to create the 2µ-based plasmid pVPS45-15. For sequence analysis, plasmid pVPS45-9 was constructed by inserting the ClaI-SacI fragment of pVPS45-10 (containing the SmaI-PvuII fragment of pVPS45-1) into the ClaI and SacI sites of pBluescript KS (Stratagene). Generation of pVPS45-6 was achieved by ligating the EcoRV-EcoRV fragment of pVPS45-1 into the SmaI site of pBluescript KS. Plasmid pVPS45-5 was constructed by inserting the BglII-BglII segment of pVPS45-1 into the BamHI site of pBluescript KS. pVPS45-21 consisted of the EcoRV-HindIII segment of pVPS45-1 inserted at the SmaI and HindIII sites of pBluescript KS. Plasmids pVPS45-4 and pVPS45-7 were generated by introducing the BglII-PvuII or SmaI-BglII fragments of pVPS45-1, respectively, into the SmaI and BamHI sites of pRS414. The integrative mapping plasmid, pVPS45-17, was constructed by inserting the ClaI-SacI fragment of pVPS45-10 into the ClaI and SacI sites of pRS304 (Sikorski and Heiter, 1989). An intermediate plasmid construction was performed in generating the vps45 deletion/disruption. Plasmid pVPS45-9 was digested with EcoRV and BglII, removing a large portion of VPS45 (see Fig. 1A), and the remainder of the vector sequences were isolated and purified. pHIS3 (from E. Phiziaky) was digested by XhoI, blunted by a Klenow fill-in reaction, and digested with BamHI. The resulting XhoI(blunt)BamHI fragment containing the HIS3 gene was isolated and ligated into the EcoRV and BglII sites of pVPS45-9 (lacking the VPS45 coding sequences) to generate disruption plasmid pVPS45-19. In order to construct a TrpE-Vps45 fusion protein, plasmid vector pATH2 (containing the trpE coding sequences; Dieckmann and Tzagoloff, 1985) was digested with SmaI and HindIII. Plasmid pVPS45-9 was digested with EcoRV and HindIII, and the 626-base fragment was purified and ligated into the SmaI and HindIII sites of the pATH2 trpE expression vector to generate pVPS45-11. Genetic and nucleic acid manipulations All standard yeast genetic procedures were adhered to as previously described (Miller, 1972; Sherman et al., 1979). Bacterial DNA transformations were performed using the protocols of Hanahan (1983). Yeast transformations followed a previously described alkali cation treatment protocol (Ito et al., 1983). Yeast strain CCY100 was generated by integrating plasmid pBHY11 (CPY-invertase::LEU2) (Horazdovsky et al., 1994) at the leu2-3,122 locus of BHY45-1. Integrative mapping studies of the cloned VPS45 gene were carried out by linearizing pVPS45-17 (VPS45, TRP1) at SphI and using the linearized plasmid to transform BHY11. Trp+ transformants (CCY101) were crossed with CCY100. Diploids were selected, sporulated, and 26 of the resulting asci were dissected. Trp+/Trp− and Vps+/Vps− segregated 2:2. All Trp+ haploid segregants also showed the Vps+ phenotype. In order to generate vps45 chromosomal deletion mutant CCY120, plasmid pVPS45-19 was digested with ScaI and SphI, and the resultant fragment containing the vps45∆2::HIS3 construct was used to transform SEY6210, SEY6211 or BHY10.5. His+ transformants of BHY10.5 were sporulated, and resultant asci were dissected. Genomic DNA from representative haploid segregants as well as from His+ SEY6210 and SEY6211 transformants was isolated and subjected to polymerase chain reaction with oligonucleotides CC451 and CC452 (see below) to confirm the presence of the appropriate deletion/disruption (Herman and Emr, 1990). The oligonucleotides CC451, 5′-GGGCGTCCGTAACGAG-3′, vps45 mutants accumulate membrane vesicles 3451 Table 1. Strains used in this study Strain S. cerevisiae SEY6210 SEY6211 BHY10.5 BHY10 BHY11 BHY45-1 CCY100 CCY101 CCY120 CCY123 CCY124 E. coli JM101 XL1-Blue Genotype Reference or source MATα leu2-3,112 ura3-52 his3-∆200 trp1-∆901 lys2-801 suc2-∆9 MATa leu2-3,112 ura3-52 his3-∆200 trp1-∆90 ade2-101 suc2-∆9 MATα/MATa leu2-3,112::pBHY11(CPY-Inv LEU2)/leu2-3,112::pBHY11(CPY-Inv LEU2) his3-∆200/his3-∆200ura3-52/ura3-52 trp1-∆901/trp1-∆901 suc2-∆9/suc2-∆9 ADE2/ade2-101 lys2-801/LYS2 SEY6210; leu2-3,112::pBHY11(CPY-Inv LEU2) SEY6211; leu2-3,112::pBHY11(CPY-Inv LEU2) SEY6210; vps45-1 BHY45-1; leu2-3,112::pBHY11(CPY-Inv LEU2) BHY11; VPS45/VPS45-TRP1 SEY6210; vps45∆2::HIS3 SEY6211; vps45∆2::HIS3 BHY10.5; vps45∆2::HIS3 Horazdovsky et al. (1994) Horazdovsky et al. (1994) This study This study This study This study This study This study ∆(lac-pro) supE thi-1 [F′ traD36 lacIq Z∆M15 proAB] supE44 thi-1 lac endA1 gyrA96 hsdR17 relA1 [F′ proAB lacIq Z∆M15 Tn10] Yanish-Perron et al. (1985) Bullock et al. (1987) Robinson et al. (1988) Robinson et al. (1988) Horazdovsky et al. (1994) and CC452, 5′-CGACTCCCCGTATCGG-3′, served as PCR primers in analyzing genomic DNA for the presence or absence of the vps45 disruption construct. Oligonucleotides CC45ST, 5′-CCTTTTTGATGTGGC-3′, CCE5, 5′-CGTTTGATTTGCAACC-3′, CC2H, 5′ATAGGCATTAAGCGG-3′, and Stratagene T3 and T7 primers were used for sequence analysis (see below). plasmid, pVPS45-11. Fusion protein production was induced, and the hybrid protein was purified by the method of Kleid et al. (1981), as modified by Herman and Emr (1990). Purified fusion protein was used to immunize New Zealand White rabbits as previously described (Horazdovsky and Emr, 1993). Collected antiserum was screened and titrated by immunoprecipitation of labeled yeast cell extracts. Cloning and sequence analysis of VPS45 BHY45-1 (vps45-1 leu2-3,112) cells carrying a plasmid encoding a CPY-invertase fusion protein (pCYI50, URA3, CEN; Johnson et al., 1987) were transformed with a plasmid-based yeast genomic DNA library (LEU2, CEN; kindly provided by Philip Hieter). Ura+/Leu+ transformants were selected, replica-plated onto YPF medium, and incubated at 30°C overnight. Vps+ colonies were identified using an overlay assay designed to detect extracellular invertase activity (Horazdovsky et al., 1994). Plasmids conferring the Vps+ phenotype were isolated (pBHY45-1) and used to transform BHY45-1 to confirm complementing activity. Various portions of the genomic sequences contained in pBHY45-1 were subcloned into the plasmid shuttle vector pRS414 (Sikorski and Heiter, 1989) and tested for the ability to complement the vps45 mutant phenotype in order to determine the minimum complementing DNA fragment shown in Fig. 1A. Exonuclease-mung bean nuclease deletions using pVPS45-9 were performed according to the pBluescript manual supplied by Stratagene, except that nuclease digestion products were size-fractionated and isolated from a 1% agarose preparative gel. Resultant nested deletion products and plasmids pVPS45-4, pVPS45-5, pVPS45-6, pVPS45-7, pVPS45-9 and pVPS45-21 were denatured and purified over 2 ml Sephacryl S-400 spun columns using the procedure described in the Pharmacia MinprepKit Plus manual. The resultant denatured singlestranded templates were hybridized to T3, T7, CC45ST, CCE5 or CC2H primers and subjected to dideoxy chain termination sequence analysis (Sanger et al., 1977) using the Sequenase sequencing protocol (US Biochemical Corp.). Protein sequences were aligned progressively, employing the method of Feng and Doolittle (1990). Cell labeling and immunoprecipitations Yeast cells were grown in SD supplemented with appropriate amino and Casamino acids (2%) to an A600 of 0.8; 5 A600 units of cells were collected by centrifugation (2,000 g for 5 minutes) and suspended in 1 ml of SD medium containing 1 mg/ml bovine serum albumin. Cells were preincubated for 10 minutes at 30°C, 50 µCi of Tran 35S-label was then added, and incubation was continued at 30°C for 10 to 30 minutes. When employed, a chase period (for the indicated duration) was initiated by addition of methionine and cysteine to final concentrations of 5 mM and 1 mM, respectively, as well as yeast extract to 0.2%; 60 minute chases were further supplemented by addition of 650 µl of SD medium containing appropriate amino acids and glucose at a final concentration of 5%. The label-chase reactions were terminated by addition of trichloroacetic acid (TCA) to a final concentration of 10%, and the precipitated proteins were analyzed by immunoprecipitation as described previously (Klionsky et al., 1988). Electron microscopy analysis SEY6210 (VPS45) and CCY120 (vps45∆2) were grown in YPD medium at 30°C to an absorbance at 600 nm (A600) of 0.5; 50 A600 units of cells were harvested by centrifugation and fixed for 1 hour at 30°C in 2 ml of 0.1 M sodium cacodylate, pH 6.8, 5 mM CaCl2, containing 3% glutaraldehyde. Cells were embedded, stained and viewed as previously described (Banta et al., 1988). CPY and PrA fractionations CPY and PrA fractionation experiments involved growth of yeast cells to an A600 of 0.8; 5 A600 units of cells were harvested by centrifugation (2,000 g for 5 minutes) and resuspended in 0.5 ml of YNB containing 1 mg/ml of bovine serum albumin, 100 µg/ml of α2macroglobulin. Cells were preincubated at 30°C for 10 minutes; 100 µCi of Tran 35S-label was added, and the cells were labeled for 10 minutes at 30°C. Chase was initiated by the addition of methionine, cysteine and yeast extract to final concentrations of 5 mM, 1 mM and 0.2%, respectively. Following a 30 minute chase period, an equal volume of ice-cold spheroplast buffer (50 mM Tris-HCl, pH 7.5, 2 M sorbitol, 40 mM NaN3, 40 mM NaF, 20 mM dithiothreitol) was added, and the cultures were incubated on ice for 10 minutes. Then 9 µg of zymolyase was added, and the cultures were incubated at 30°C for 30 minutes. The cultures were then centrifuged at 13,000 g for 1 minute to generate an intracellular (I) pellet fraction and an extracellular (E) media fraction. The presence of CPY and PrA proteins in each fraction was determined by immunoprecipitation (Klionsky et al., 1988; Robinson et al., 1988). Preparation of antiserum Bacterial cells JM101 were transformed with a trpE-VPS45 gene fusion Subcellular fractionations SEY6210 cells or CCY120 cells harboring plasmid pVPS45-15 were 3452 C. R. Cowles, S. D. Emr and B. F. Horazdovsky A S E E H B B Sp X P 0.5 kb VPS45 S E S E E H B B Sp B Sp HIS3 B WT vps45∆2 VPS45 vps45∆2 Fig. 2. Nucleotide sequence of VPS45 and amino acid sequence comparison of Vps45p with Sec1p homologues. (A) The nucleotide sequence and the deduced amino acid sequence of VPS45 are shown. The sequence accession number for VPS45 is U11049. (B) Vps45p and the S. cerevisiae Sec1p homologues Sec1p (Aalto et al., 1991), Sly1p (Dascher et al., 1991) and Vps33p (Banta et al., 1990) were aligned progressively (Feng and Doolittle, 1990). Regions of amino acid identity are shown in black boxes. The putative leucine zipper motif of Vps45p is indicated by asterisks and a region of high homology corresponding to residues 226 to 248 of Vps45p is boxed. minutes at 30°C and chased for 30 minutes as described above. Spheroplasts were harvested at 500 g and resuspended in ice-cold lysis buffer (50 mM Tris-HCl, 200 mM Sorbitol, 1 mM EDTA; Horazdovsky and Emr, 1993). The resultant cell suspension was Dounced six times in an ice-cold tissue homogenizer, and then subjected to sequential centrifugation at 500 g (10 minutes), 13,000 g (10 minutes) and 100,000 g (60 minutes) as described previously (Horazdovsky and Emr, 1993). To determine the nature of Vps45p-P100 association, equal samples of the cleared lysate (500 g supernatant) were adjusted to 2 M urea, 1% Triton X-100, 1 M NaCl, or were left untreated. All samples were incubated on ice for 10 minutes, then subjected to a 100,000 g centrifugation for 1 hour. The level of Vps45p, ALP, glucose-6-phosphate dehydrogenase, Kex2p or PM ATPase in each fraction was determined by immunoprecipitation as previously described (Horazdovsky and Emr, 1993). RESULTS 25˚C WT vps45∆2 VPS45 vps45∆2 38˚C Fig. 1. Characterization and disruption of the VPS45 locus. (A) A restriction map of the genomic DNA fragment containing the 3.5 kb SmaI-PvuII complementing fragment. The VPS45 coding sequence is represented by the black arrow; S, SmaI; E, EcoRV; H, HindIII; B, BglII; Sp, SphI; X, XhoI and P, PvuII. The 1.6 kb EcoRV-BglII fragment containing VPS45 sequence was replaced by the HIS3 gene (open arrow) to generate the deletion/disruption strain CCY120 (vps45∆2). (B) Growth phenotypes of wild-type cells (SEY6210, WT), cells carrying the vps45 null mutation (vps45∆2, CCY120), or vps45∆2 cells containing the complementing plasmid pVPS45-10 (VPS45) on YPD plates are shown following three days of incubation at either 25 or 38°C. No significant difference in growth rates was observed between 25 and 30˚C for all strains shown. grown at 30°C to an A600 of 0.8 in SD supplemented with appropriate amino acids and Casamino acids (2%). Cells were collected by centrifugation (2,000 g for 5 minutes), and spheroplasts were generated as previously described (Vida et al., 1990). Spheroplasts were incubated for 10 minutes at 30°C prior to addition of 30 µCi of Tran 35S-label per A600 unit of cells. Spheroplasts were labeled for 30 Cloning and sequencing of the VPS45 gene vps45 mutants were isolated using a hybrid-protein-based selection scheme (Bankaitis et al., 1986; Robinson et al., 1988). The hybrid protein consisted of a N-terminal portion of vacuolar proteinase A (PrA) fused to the normally secreted enzyme invertase. In wild-type cells, the vacuolar protein sorting signal in the PrA portion of the fusion protein directed it to the vacuole (PrA-Inv 137) (Klionsky et al., 1988). Using a yeast strain deleted for the SUC2 gene encoding endogenous invertase (SEY6210), vps mutants that missort and secrete the PrA-invertase hybrid were selected on the basis of their ability to utilize sucrose, an invertase substrate, as their sole carbon source. This approach identified many spontaneous vps mutant strains, including vps45. One vps45 mutant allele, vps45-1, exhibited a temperature-conditional (ts) growth phenotype, which was employed to clone the wild-type gene affected in this mutant. BHY45-1 (vps45-1) cells were transformed with a YEp24 (URA3, 2µ)-based genomic library (Carlson and Botstein, 1982). Approximately 16,000 of the resultant Ura+ transformants were replica-plated and grown at 38°C. Seven of the Ura+ transformants grew at the elevated temperature. The genomic library plasmid from one of these transformants was isolated, amplified in E. coli, and used to retransform BHY45-1. The plasmid was found to confer temperature resistance to the transformants, and restriction enzyme mapping revealed that the complementing library plasmid contained an approximately 7 kb genomic DNA insert. The complementing activity was further refined and found to be contained on a 3.5 kb genomic DNA fragment (Fig. 1A) that was capable of complementing both the vps45-1 ts growth phenotype as well as the vacuolar protein sorting defect when present on the low copy plasmid pVPS45-10 (see below). Integrative mapping studies were used to demonstrate that the cloned genomic vps45 mutants accumulate membrane vesicles 3453 A 1 37 73 109 145 181 217 253 289 325 361 397 433 469 505 541 577 B CCCGGGCGTCCGTAACG AGTACTGACTGTATGGCGAAAAGTTGCCAGAAATGTTCATTTTTGTTTCTGCCAATTTCAGGAAAGGGTAACGTCATTACAGTTAGTTAACGTTCGAACTTACTTATA GAAGTGCCTTGGCTCATATGCAATGTACCCAGAGTAACATTAAGGAGTGAAGAGGTACAGTGACTTGGTTTTGAGTTAAGGCCATCTTTTACTGTATAGAACAAAGAA ATGAACCTTTTTGATGTGGCTGACTTTTATATAAACAAAATTGTGACTTCCCAATCGAAATTGAGCGTAGCCAATGTCAATGAACACCAAAGGATTAAGGTTTTGCTG M N L F D V A D F Y I N K I V T S Q S K L S V A N V N E H Q R I K V L L TTGGATAAGAATACCACACCTACGATATCCTTATGTGCCACTCAAAGTGAGTTGTTGAAGCATGAAATATATCTGGTAGAAAGAATAGAAAATGAGCAACGTGAAGTG L D K N T T P T I S L C A T Q S E L L K H E I Y L V E R I E N E Q R E V TCCAGGCATTTAAGGTGCTTAGTTTACGTTAAACCCACAGAGGAAACACTGCAACATCTGCTGCGTGAGTTAAGAAATCCTCGGTACGGCGAGTATCAAATATTCTTT S R H L R C L V Y V K P T E E T L Q H L L R E L R N P R Y G E Y Q I F F AGTAATATTGTCTCTAAATCTCAATTAGAACGGCTAGCTGAATCTGACGACTTGGAAGCTGTTACTAAGGTGGAAGAAATATTCCAAGACTTTTTTATATTAAACCAA S N I V S K S Q L E R L A E S D D L E A V T K V E E I F Q D F F I L N Q GATTTATTTTCGTTTGATTTGCAACCAAGAGAATTTTTAAGTAATAAATTGGTTTGGAGCGAAGGGGGGCTAACAAAATGTACCAACAGCTTAGTTTCTGTGCTTTTA D L F S F D L Q P R E F L S N K L V W S E G G L T K C T N S L V S V L L TCCTTAAAGATAAAACCAGATATCAGGTATGAAGGAGCAAGTAAAATTTGTGAAAGATTGGCTAAAGAAGTTTCCTATGAGATTGGTAAAAACGAAAGAACTTTTTTT S L K I K P D I R Y E G A S K I C E R L A K E V S Y E I G K N E R T F F GATTTTCCTGTGATGGATTCGACACCTGTGTTACTAATTTTAGATCGTAATACTGATCCTATAACACCTTTACTTCAACCTTGGACCTACCAATCAATGATCAATGAG D F P V M D S T P V L L I L D R N T D P I T P L L Q P W T Y Q S M I N E TATATAGGCATTAAGCGGAATATAGTTGATTTATCGAAAGTGCCTAGAATTGATAAAGACCTGGAGAAGGTCACCTTATCATCAAAGCAAGATGCTTTCTTCAGGGAT Y I G I K R N I V D L S K V P R I D K D L E K V T L S S K Q D A F F R D ACCATGTATTTGAATTTTGGTGAATTGGGTGATAAAGTAAAACAATATGTGACTACATACAAAGACAAGACACAAACCAACAGCCAAATAAATTCCATTGAGGATATT T M Y L N F G E L G D K V K Q Y V T T Y K D K T Q T N S Q I N S I E D I AAAAACTTTATTGAGAAGTATCCAGAGTTTAGAAAATTATCTGGAAATGTTGCAAAGCATATGGCTATAGTGGGGGAATTAGACAGACAGTTGAAGATAAAAAATATA K N F I E K Y P E F R K L S G N V A K H M A I V G E L D R Q L K I K N I TGGGAAATTAGTGAAATAGAACAAAATCTATCAGCACACGATGCCAATGAAGAAGATTTCTCCGATTTGATTAAATTGCTACAAAATGAAGCAGTTGATAAGTATTAC W E I S E I E Q N L S A H D A N E E D F S D L I K L L Q N E A V D K Y Y AAGTTAAAGCTTGCATGTATTTATTCTTTAAACAATCAAACCAGCTCAGACAAAATCCGTCAACTAGTTGAGATTCTGTCTCAACAACTTCCTCCAGAGGACGTCAAC K L K L A C I Y S L N N Q T S S D K I R Q L V E I L S Q Q L P P E D V N TTTTTCCATAAATTTAAATCGCTTTTTAGCCGCCAGGATAAAATGACTCAAAGTAACCATGACAAGGACGATATATTAACCGAACTAGCAAGAAGATTTAATAGTAGA F F H K F K S L F S R Q D K M T Q S N H D K D D I L T E L A R R F N S R ATGAATTCTAAGAGCAACACCGCTGAAAACGTCTATATGCAACATATTCCGGAAATTTCGTCATTACTAACAGATCTCTCTAAAAATGCGTTATTCAGGGATCGTTTC M N S K S N T A E N V Y M Q H I P E I S S L L T D L S K N A L F R D R F AAAGAAATAGATACTCAAGGCCATAGAGTGATCGGAAACCAGCAGAGCAAAGATATTCCTCAGGATGTAATATTGTTTGTTATTGGCGGTGTAACTTATGAGGAGGCA K E I D T Q G H R V I G N Q Q S K D I P Q D V I L F V I G G V T Y E E A AGGCTAGTCCATGATTTCAATGGAACGATGAATAACAGAATGAGGGTGGTTTTAGGAGGCACCTCTATACTTTCAACTAAAGAATATATGGATTCTATTAGATCTGCA R L V H D F N G T M N N R M R V V L G G T S I L S T K E Y M D S I R S A AAATAAATAAGGATTATCTTATTCTAAAATTCTATTTTATATATGAGGCATAAATCTATATAACTTTTTCGATGCAGATAAAACATTTTACTATTGCCAAGCGAAATT K * (577) AGGTTCTCATTTTCTTCATTCGGTGCCTAATAATTGCAACTTTGTTCGGTGATCCTTCTATGTGCATGCGTCATAATAACTTAACTGGAAAAAACTTTCTCAACTTAC GACAAAAAACTCCGATACGGGGAGTCGAACCCCGGTCTCCACGGTGAAAGCGTGATGTGATAGCCGTTACACTATATCGGACAATAATTGTTGGAAATTCATTACAAA GGTAAATTACTATATGGAAACTTTAC 17 125 233 341 449 557 665 773 881 989 1097 1205 1313 1421 1529 1637 1745 1853 1961 2069 2177 2285 2311 3454 C. R. Cowles, S. D. Emr and B. F. Horazdovsky fragment corresponded to the VPS45 locus (see Materials and Methods). Within the 3.5 kb VPS45 complementing fragment, a single large open reading frame was identified and predicted to code for a protein of 67,012 Da (Fig. 2A). Hydropathy analysis (Kyte and Doolittle, 1982) predicted the gene product to be largely hydrophilic and to lack hydrophobic N-terminal signal sequences and potential transmembrane domains. One predicted structure found within the open reading frame was a putative leucine zipper motif, composed of four leucine residues spaced at seven-residue intervals (Fig. 2B). Comparison of the deduced Vps45p amino acid sequence with known proteins currently available in the EMBL and GenBank data bases also revealed a similarity between Vps45p and the Sec1 protein family; members of this family are thought to be involved in vesicle targeting and/or fusion events (Novick et al., 1980; Ossig et al., 1991; Gengyo-Ando et al., 1993; Hata et al., 1993; Pelham, 1993; Garcia et al., 1994; Pevsner et al., 1994). Progressive alignments (Feng and Doolittle, 1990) of the Vps45p sequence with several members of the Sec1 protein family were performed, and Vps45p was found to share the greatest sequence identity with the S. cerevisiae Sly1, Sec1 and Vps33 proteins of the Sec1 family (Fig. 2B) (Banta et al., 1990; Wada et al., 1990; Aalto et al., 1991; Dascher et al., 1991). All members of this group share low but significant levels of sequence identity. Notably, a region of 50% or greater identity among these four gene products is observed within the span of Vps45p amino acid residues 226-248 (Fig. 2B, boxed region). The role this sequence plays in Vps45p function is not yet known. A portion of the previously identified SOE1 coding sequence (Su et al., 1990) was also discovered adjacent to the VPS45 open reading frame, localizing the VPS45 gene to chromosome VII. Disruption of the VPS45 gene To determine the phenotypic consequences resulting from loss of VPS45 gene product function, a vps45 deletion/disruption allele was constructed. The EcoRV-BglII fragment of VPS45 was replaced with a DNA fragment containing the HIS3 gene (Fig. 1A). The DNA segment containing the deletion/disruption construct was then used to transform a wild-type diploid strain (BHY10.5). His+ diploid transformants were selected and sporulated. All spores were viable, indicating that VPS45 was not essential for growth. In addition, analysis of the resultant tetrads showed that His+/His− and Vps+/Vps− phenotypes segregated 2:2, with all His+ segregants being Vps−. However, strains carrying the vps45 null mutation did exhibit a temperature-conditional growth defect. Unlike wild-type cells, ∆vps45 cells were unable to grow at 38°C (Fig. 1B). This temperature-conditional phenotype was completely complemented by the presence of the cloned VPS45 gene on a low copy plasmid vector (Fig. 1B). These results indicated that while Vps45p was not required for vegetative growth at permissive temperatures (25°C), Vps45p was required for growth at elevated temperatures. Presumably vacuole functions compromised in the vps45 mutant were essential for growth at 38˚C. Vacuolar proteins undergo compartmental-specific modification as they transit through the secretory pathway en route to the vacuole (Stevens et al., 1982). Many vacuolar proteins are modified by core oligosaccharides when they enter the ER. A VPS45 vps45∆2 I E I E 1 2 3 4 vps45∆2 VPS45 I E p2CPY mCPY B 5 6 VPS45 vps45∆2 I E I E proPrA mPrA 1 2 3 4 Fig. 3. Intracellular sorting of vacuolar hydrolases. Wild-type cells (VPS45, SEY6210), cells containing the vps45 null allele (vps45∆2, CCY120) and CCY120 transformed with the complementing plasmid pVPS45-10 (vps45∆2/VPS45) were labeled for 10 minutes at 30°C with Tran 35S-label, chased for 30 minutes at 30°C, then converted to spheroplasts. The labeled cultures were separated into spheroplast (internal, I) and media (external, E) fractions. The presence of (A) CPY and (B) PrA in these fractions was determined by immunoprecipitation. The migration positions of Golgi-modified precursors (p2CPY, proPrA) and mature (mCPY, mPrA) proteins are shown. These core oligosaccharides are then extended by the addition of mannose residues in subsequent Golgi compartments. This leads to an easily identifiable Golgi-modified protein precursor. In the case of carboxypeptidase Y (CPY), this precursor is referred to as p2CPY (69 kDa); and in the case of PrA, proPrA (48 kDa). In wild-type cells, when these proteins reach the vacuole, the pro segments of p2CPY and proPrA are removed, resulting in the mature vacuolar forms of the enzymes, mCPY and mPrA (61 kDa and 42 kDa, respectively). We examined the vacuolar protein sorting capacity of wild-type (SEY6210), vps45∆2 and vps45∆2 cells carrying the cloned VPS45 gene. Cells were labeled for 10 minutes with Tran 35S-label ([35S]methionine and [35S]cysteine) and then chased for 30 minutes by the addition of unlabeled methionine and cysteine. The pulse-labeled cells were treated with zymolyase to remove their cell walls, and the resultant spheroplasts were separated from the culture medium by centrifugation. The presence of CPY and PrA in the spheroplast pellet (I, internal) and media (E, external) fractions was determined by immunoprecipitation. As expected, wild-type (VPS45) cells properly delivered CPY and PrA to the vacuole as evidenced by the presence of these proteins in the internal cell fractions as their mature vacuolar forms (mCPY and mPrA; Fig. 3A,B, lane 1). vps45∆2 cells transformed with pVPS45-10 (VPS45) also completely matured CPY (Fig. 3A, lane 5). In contrast, the vast majority of CPY and PrA were found as their Golgi-modified precursor forms (p2CPY, proPrA) in vps45∆2 cells. Approximately 85% of the p2CPY was secreted from these cells (Fig. 3A, lane 4). vps45 mutants accumulate membrane vesicles 3455 Fig. 4. Morphology of the vps45∆2 mutant strain. Wild-type cells (A, SEY6210) or cells containing the vps45 null allele (B and C, CCY120) were prepared for electron microscopic analysis. In A, n identifies the nucleus; v, vacuole. Bars in A and B, 0.5 µm. The broken line box region in B is enlarged in C to show a cluster of 4050 nm membrane vesicles. Bar in C, 0.1 µm. shown). These results indicated that Vps45p function is required for the delivery of soluble vacuolar proteins (CPY and PrA). Vesicular structures are accumulated in vps45∆2 cells The effect of deleting VPS45 on vacuole morphology and other cellular structures was analyzed using both light and electron microscopy. Vacuole morphology was first analyzed in cells grown at 30°C using the fluorescent vacuole-specific vital stain 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDCFDA). In contrast to wild-type cells, that contained two to five vacuole structures per cell, the vast majority of vps45∆2 cells contained a single large vacuole structure (data not shown). Many of the vps45∆2 cells also accumulated CDCFDA in much smaller structures (~10 per cell) found throughout the cytoplasm (data not shown). Approximately 60% of newly formed buds in vps45∆2 cells contained a vacuole compartment. This was in contrast with budded wild-type cells, where greater than 90% of the buds contained a vacuole compartment. These observations are consistent with the previous assignment of vps45 mutants to the class D mutant class (Raymond et al., 1992). Examination of vps45∆2 mutant cells by electron microscopy uncovered a number of alterations in subcellular structure. Most noticeably, in 5 to 10% of random vps45∆2 cell sections, large clusters of 40-50 nm vesicles were observed (Fig. 4B,C; if vesicle clusters are assumed to be spherical and of uniform size, we would anticipate that 40-60% of cells in the vps45∆2 population possessed a vesicle cluster). The vesicles contained within these clusters were evenly spaced at approximately 20 nm and the majority of the clusters themselves were found adjacent to the vacuole. Such vesicle clusters were not observed in wild-type cells (Fig. 4A) nor in other class D vps mutants (e.g. vps21, data not shown). In addition, vps45∆2 cells were seen to accumulate 40-50 nm membrane vesicles in cell sections lacking observable clusters. These single vesicles were quantitated and found to be present at levels approximately four-fold higher than those observed in wild-type cells (based on comparison of the number of vesicles/µm2 of cytosol in greater than 100 cell sections for both mutant and wild-type cells). ProPrA was also secreted from vps45∆2 cells, although a significant portion (65%) was retained inside the cells as well (Fig. 3B, lanes 3 and 4). This retained proPrA did not mature when chase times were extended to 90 minutes (data not Identification and subcellular localization of Vps45p A TrpE-Vps45 fusion protein (containing Vps45p residues 187 to 398, see Fig. 2A) was used to raise polyclonal antiserum that recognized Vps45p. Using this antiserum in immunoprecipitation experiments, a single protein migrating with an apparent molecular mass of 67 kDa was detected in labeled wild-type yeast cell extracts (SEY6210; WT, Fig. 5, lane 2). The mass of this protein species was in good agreement with the predicted mass of Vps45p, based on the sequence analysis. This protein was not detected in cells lacking the VPS45 coding sequence (vps45∆2; ∆, Fig. 5, lane 1) nor was Vps45p detected in labeled wild-type cell extracts when preimmune serum was 3456 C. R. Cowles, S. D. Emr and B. F. Horazdovsky Chase: ∆ 0' WT 0' 60' Table 2. Subcellular distribution of Vps45p and marker proteins 2µ45 0' 60' Vps45p 67 kDa Protein P13 S100 P100 Vps45p (single copy) 10* 25 65 Vps45p (multi-copy) 5 90 5 Alkaline phosphatase (vacuole membrane) 80 15 5 PM ATPase (plasma membrane) 80 10 10 Kex2p (Golgi membrane) 5 15 80 Glucose-6-P dehydrogenase (cytosol) 0 100 0 *Values are percentages. 1 2 3 4 5 Fig. 5. Immunoprecipitation of the VPS45 gene product. Wild-type cells (WT; SEY6210, lanes 2 and 3), cells containing the vps45 null allele (∆; CCY120, lane 1), or CCY120 carrying the 2µ expression plasmid pVPS45-15 (2µ45; lanes 4 and 5) were labeled for 10 minutes with Tran 35S-label at 30°C. Labeling was terminated by addition of TCA (10%; lanes 1, 2 and 4) or a 60 minute chase period at 30°C was included prior to TCA precipitation (lanes 3 and 5). The TCA-precipitated proteins were processed for immunoprecipitation using Vps45p antiserum, and the antigen-antibody complexes were resolved by SDS-PAGE and fluorography. The size of Vps45p was determined by comparison to molecular mass standards. The amount of material loaded in lanes 4 and 5 represents one-fifth of that of the material loaded in lanes 1-3; additionally, the exposure time for lanes 4 and 5 was approximately one-half of that for lanes 1-3. used (data not shown). Vps45p also appeared to be stable for at least 60 minutes, as no loss of Vps45p signal was detected after a 60 minute chase period in wild-type cells (Fig. 5, lane 3). In addition, when present on a multicopy plasmid (2µVPS45), Vps45p was stably expressed at levels approximately 20-fold higher than those seen in cells containing a single copy of VPS45 (2µ45; Fig. 5, lanes 4 and 5; the amount of material and exposure time of the panel containing lanes 4 and 5 are one-fifth and one-half those of the panel containing lanes 1-3, respectively). In wild-type cells, the expression level of Vps45p was approximately one-tenth that observed for CPY, suggesting a Vps45p abundance of ~0.01% of total cell protein. Subcellular fractionation was performed to help determine the intracellular location of Vps45p. Spheroplasts generated from wild-type cells were labeled with Tran 35S-label for 30 minutes, chased for 30 minutes, lysed, and the spheroplast lysate was subjected to a set of sequential centrifugations. Following a 500 g centrifugation to remove unlysed spheroplasts, the cleared lysate was centrifuged at 13,000 g, generat- ing a supernatant (S13) and pellet (P13) fraction. The S13 was then centrifuged at 100,000 g to generate a second set of supernatant (S100) and pellet (P100) fractions. The presence of Vps45p as well as organelle marker proteins in each of these fractions was determined by immunoprecipitation. The majority of Vps45p (65%) was found in the high-speed pellet (P100) and thus cofractionated with the Golgi membrane marker Kex2p (Table 2). While this result suggested that Vps45p may directly associate with Golgi membranes, we cannot rule out the possibility that Vps45p may also associate with an endosomal compartment or intermediate vacuolar transport vesicles, as no markers for these structures are yet available. In addition, a portion of Vps45p was found in a S100 (Fig. 6A, lane 3; Table 2) fraction; and a small amount of Vps45p was associated with the dense membrane fraction, P13 (Fig. 6A, lane 2; Table 2). Markers for the vacuole, ER, plasma membrane, and mitochondria are enriched in this P13 fraction (Table 2) (Marcusson et al., 1994). Twentyfold overexpression of Vps45p resulted in a markedly different subcellular fractionation pattern of Vps45p: the vast majority of overexpressed Vps45p was found in a S100 fraction (Table 2). This result was in contrast to the predominant localization of wild-type levels of Vps45p in a P100 fraction and suggested that overexpression of Vps45p saturates an interaction site located in the P100 fraction. To determine the nature of the association of Vps45p with these particulate cell fractions, cleared labeled cell lysates were treated with a number of reagents prior to centrifugation at 100,000 g. When the lysate was pretreated with detergent (1% Triton) or 2 M urea, Vps45p was released into a soluble cell fraction (Fig. 6B, lanes 3-6). In contrast, when cell lysates were treated with 1 M NaCl, the fractionation pattern of Vps45p was unchanged, with 70% of Vps45p pelleting in a P100 fraction (Fig. 6B, lanes 7 and 8). This result suggested that Vps45p is peripherally associated with a membrane, possibly via hydrophobic (salt-stable) protein-protein interactions. DISCUSSION Vps45p is required for the efficient sorting of proteins to the yeast vacuole. Cells that lack the VPS45 gene product missort vps45 mutants accumulate membrane vesicles 3457 A S13 P13 S100 P100 Vps45p 1 2 3 4 B No Wash S P 1% Triton S P 2M Urea S P 1M NaCl S P 1 3 5 7 Vps45p 2 4 6 8 Fig. 6. Subcellular fractionation of the VPS45 gene product. (A) Wild-type cells were converted to spheroplasts, labeled with Tran 35S-label for 30 minutes, and chased for 30 minutes. The spheroplasts were lysed, and the lysate was cleared of unbroken spheroplasts. Initial fractionations were performed by centrifugation at 13,000 g, yielding supernatant (S13, lane 1) and pellet (P13, lane 2) fractions. The S13 fraction was then subjected to a second centrifugation at 100,000 g, resulting in a supernatant (S100, lane 3) and a pellet fraction (P100, lane 4). Vps45p, as well as various marker proteins (Table 2), was quantitatively immunoprecipitated from each fraction. (B) Cleared cell lysate was pretreated with buffer alone (lanes 1 and 2), 1% Triton X-100 (lanes 3 and 4), 2 M urea (lanes 5 and 6), or 1 M NaCl (lanes 7 and 8) prior to centrifugation at 100,000 g. The presence of Vps45p in the resultant supernatant (S) and pellet (P) fractions was determined by quantitative immunoprecipitation. and secrete soluble vacuolar proteins in their Golgi-modified precursor form. In addition, ∆vps45 mutant cells show a temperature-sensitive growth defect and accumulate clusters of small membrane vesicles. Comparison of the predicted Vps45p amino acid sequence with other known proteins revealed that Vps45p is a member of the Sec1 protein family. Members of this family have been implicated in vesicle targeting in a variety of intercompartmental protein transport events. The VPS45 gene product seems to serve a similar function in the vacuolar protein localization pathway. A current model of vesicle targeting and fusion proposes that the specificity required to accurately target vesicles derived from a donor organelle to a distinct acceptor organelle is mediated by a set of membrane proteins generically referred to as SNARE proteins (Söllner et al., 1993a,b). According to this model, vesicles fuse with their appropriate target following interaction of a vesicle-SNARE (v-SNARE) with its cognate target-SNARE (t-SNARE) on the acceptor organelle. The SNARE protein family is typified by the well-characterized synaptic vesicle VAMP/synaptobrevin v-SNAREs (Trimble et al., 1988; Baumert et al., 1989) and the plasma membrane syntaxin (Bennett et al., 1992) and SNAP-25 (Oyler et al., 1989) t-SNAREs. VAMP has been shown to interact specifically with syntaxin (Calakos et al., 1994), in a complex that also includes SNAP-25 (Söllner et al., 1993a). It is thought that this recognition event docks synaptic vesicles at the plasma membrane. Following docking, the vesicles fuse with the plasma membrane, releasing their contents. Synaptic vesicle recognition and fusion appear to be regulated (Bennett and Scheller, 1993), and one candidate regulator of this process is a member of the Sec1 protein family, n-Sec1. Recent studies have shown that n-Sec1 from mammalian neuronal cells specifically interacts with syntaxin (Hata et al., 1993; Garcia et al., 1994; Pevsner et al., 1994), and it has been proposed that n-Sec1 might function to regulate formation of the vesicle docking complex (Pevsner et al., 1994). In yeast, sec1 ts mutant cells accumulate 100 nm Golgi-derived secretory vesicles, suggesting a role for Sec1p in targeting and/or fusion of secretory vesicles with the plasma membrane (Novick et al., 1980). Interestingly, two multicopy suppressors of a sec1 ts mutant (SSO1 and SSO2) have been identified, and their predicted gene products share significant sequence homology with mammalian syntaxin (Aalto et al., 1993). These observations indicate that, like n-Sec1 in mammalian cells, yeast Sec1p may be interacting with Sso1p and Sso2p to facilitate vesicle docking at the plasma membrane. In addition to Vps45p, other yeast Sec1p family members include Sly1p and Vps33p. Sly1p has been implicated in targeting and/or fusion of ER-derived vesicles with the Golgi (Dascher et al., 1991; Ossig et al., 1991). Vps33p is thought to function in protein delivery (via vesicle transport intermediates) to the vacuole (Banta et al., 1990; Wada et al., 1990). Although vps33 and vps45 mutants are both defective for delivery of vacuolar hydrolases, their vacuole morphologies are strikingly different. In contrast to the enlarged vacuoles of vps45 mutants, vps33 mutants lack vacuoles (and are therefore designated class C). The subcellular fractionation profile of Vps33p also markedly differs from the one we observe for Vps45p (Banta et al., 1990; Fig. 6A). These data suggest separate sites of action for Vps33p and Vps45p. It is possible that one of these proteins may function in Golgi-to-endosome protein transport while the other plays a role in an endosome-to-vacuole protein delivery event. Our present data suggest that Vps45p functions in a Golgi-toendosome delivery event (see below). The accumulation of 40-50 nm vesicles in vps45 deletion mutants (Fig. 4B,C) indicates that like other members of the Sec1 protein family, Vps45p functions at a vesicle docking and/or fusion event. These vesicles are likely transport intermediates of the vacuolar protein sorting pathway. Two forms of vesicles are found in vps45 mutants, those that are apparently free in the cytoplasm and those that are found in clusters. The functional significance of the vesicle clustering is unclear, but it may result from a homotypic interaction that occurs prior to fusion with the target organelle and is exaggerated in vps45 mutant cells. It should be noted that the clustering of vesicles in ∆vps45 cells does not appear to be a fixation artifact, as other class D vps mutant cells that accumulate similar vesicles (vps21) (Horazdovsky et al., 1994) do not contain these clusters. In addition, the vesicles within each cluster are regularly spaced at ~20 nm. This spacing may represent the 3458 C. R. Cowles, S. D. Emr and B. F. Horazdovsky area occupied by a vesicle coat (10 nm on each vesicle). Since the staining procedure used here for electron microscopic analysis highlights cell membranes, other staining techniques will be needed to test for the presence of protein in this 20 nm space. Interestingly, most of the vesicle clusters observed in ∆vps45 cells are found in association with the vacuole. It is unclear if this association is meaningful, as the vacuole occupies a large portion of cell volume, and there is a reasonably high probability that clusters would be randomly found near vacuoles. Furthermore, the fact that vps45 mutant cells contain a vacuole, suggests that vacuolar membrane constituents are delivered independently of Vps45p function. Vacuolar proteins move from the Golgi to the vacuole by transiting through a prevacuolar endosome-like compartment (Vida et al., 1993). The movement of vacuolar proteins from the Golgi to the endosome is also thought to involve vesicular transport intermediates (Horazdovsky et al., 1994; Marcusson et al., 1994). Vps45p function may be required for these earlier vesicle docking/fusion events. The fractionation pattern of Vps45p is consistent with this assignment, as both the endosomal compartment and Vps45p are enriched in a highspeed membrane pellet fraction (P100) (Fig. 6A) (Vida et al., 1993). Very little Vps45p is found in a lower-speed membrane pellet (P13) that is highly enriched in vacuolar membranes. Further localization studies will be required to determine the precise subcellular location of Vps45p. The peripheral association of Vps45p with cellular membranes (Fig. 6) and the observation that this association can be saturated (Table 2) suggest that Vps45p may be interacting with a specific and limiting membrane component. A reasonable candidate for this membrane component would be a member of the syntaxin protein family. A syntaxin homolog that functions in the vacuolar protein localization pathway has been discovered recently and is encoded by the PEP12 gene (K. A. Becherer and E. W. Jones, personal communication). Interestingly, pep12 mutants fall into the class D vps morphology group and share similar vacuolar protein sorting and conditional growth phenotypes with vps45 mutants (unpublished results) (Raymond et al., 1992). Like n-Sec1 binding to syntaxin in neuronal cells, Vps45p may bind to Pep12p to facilitate vesicle-mediated vacuolar protein transport. Though the exact mechanism by which these proteins facilitate vesicle transport is unclear, Vps45p could serve a number of functions in a vesicle docking and/or fusion event. Vps45p may act to stabilize the interaction of a vacuolar sorting pathway t-SNARE, presumably Pep12p, with a corresponding v-SNARE, and thereby facilitate the vesicle fusion event. In the absence of Vps45p, the SNARE complex may rapidly dissociate and vesicle fusion may be hindered, resulting in accumulation of small vesicles. Alternatively, Vps45p may play a direct role in vesicle recognition of the appropriate target membrane, as there appears to be a different Sec1p family member associated with each type of vesicle-targeting event that has been described. Finally, Vps45p may functionally interact with a rab-like GTPase, and together these proteins may facilitate vesicle targeting and/or fusion. VPS21 codes for a small GTP-binding protein (Horazdovsky et al., 1994; Singer-Krüger et al., 1994), and vps21 mutants share many phenotypes with vps45 mutants, most notably the accumulation of 40-50 nm vesicles (Horazdovsky et al., 1994). Potential functional associations between Sec1 protein family members (Sec1p and Sly1p) and other rab-like GTP-binding proteins (Sec4p and Ypt1p) have been noted in other yeast vesicletargeting events (Bruno et al., 1988; Segev et al., 1988; Dascher et al., 1991). A similar functional interaction between Vps21p and Vps45p may be involved in the vacuolar protein delivery pathway; however, such an interaction has not been identified. Both genetic and biochemical approaches are in progress to test directly for physical interactions between Vps45p and other components of the vacuolar protein sorting pathway (i.e. Vps21p, Pep12p). In addition, the functional significance of the leucine zipper motif and those domains of Vps45p most highly conserved with other Sec1p family members is being examined. These studies should help define a more precise role for Vps45p in vacuolar protein sorting as well as provide insights into the general role that Sec1p family members play in vesicle trafficking. We thank the members of the Emr laboratory and Marylin Farquhar for helpful input during the course of this work. We also gratefully thank Daniel Szeto for cloning VPS45; Russell Doolittle for his help in progressive sequence alignment; Michael McCaffery and Tammie McQuistan for their outstanding electron microscopy work; and Bill Wickner and Randy Schekman for generously supplying Kex2p and PM ATPase antisera. This work was supported by a grant from the National Institutes of Health (GM-32703) and the National Cancer Institute (CA58689). C.R.C. is a member of the Biomedical Sciences Graduate Program and a Lucille P. 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