Mutations in the VPS45 Gene, a SEC1 Homologue, Result in Vacuolar Protein Sorting Defects and Accumulation of Membrane Vesicles

Journal of Cell Science 107, 3449-3459 (1994) 3449 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 Saccha- kDa protein in labeled yeast cell extracts. Subcellular frac- romyces cerevisiae have uncovered a large number of tionation studies demonstrate that the majority of Vps45p mutants (vps) that missort and secrete vacuolar hydrolases. is associated with a high-speed membrane pellet fraction A small subset of vps mutants exhibit a temperature-con- that includes Golgi, transport vesicles and, potentially, ditional growth phenotype and show a severe defect in the endosomal membranes. Significantly, this fraction lacks localization of soluble vacuolar proteins, yet maintain a ER, vacuole and plasma membranes. Overexpression of near-normal vacuole structure. Here, we report on the Vps45p saturates the sites with which Vps45p associates. A cloning and characterization of the gene affected in one of vps45 null mutant accumulates vesicles, many of which these mutants, VPS45, which has been found to encode a were found to be present in large clusters. This accumula- member of a protein family that includes the yeast proteins tion of potential transport vesicles indicates that Vps45p Sec1p, Sly1p and Vps33p, as well as n-Sec1, UNC18 and may facilitate the targeting and/or fusion of these vesicles Rop from other eukaryotic organisms. These proteins are in the vacuolar protein sorting pathway. thought to participate in vesicle-mediated protein transport events. Polyclonal antiserum raised against a TrpE-Vps45 fusion protein specifically detects a stable 67 Key words: VPS45, SEC1, vacuole, protein sorting, transport vesicle INTRODUCTION fragmented, while class C mutants lack any identifiable vacuole structures. Class D vps mutants are morphologically The localization of proteins to the lysosome-like vacuole of similar to class A strains but have a large, single vacuolar Saccharomyces cerevisiae is a complex process and involves structure and exhibit defects in mother-to-daughter cell several distinct targeting and delivery events. Like secreted vacuolar inheritance. Class E mutants contain a novel proteins, vacuolar proteins are first translocated into the endo- endosome-like compartment in addition to normal vacuoles, plasmic reticulum (ER), and subsequently transit to and and class F mutants contain a single large vacuole structure through the compartments of the Golgi complex (Stevens et that is encircled by smaller, fragmented vacuoles. Even though al., 1982). In a late Golgi compartment, vacuolar proteins are some vps mutants exhibit severe defects in vacuolar morphol- then actively sorted away from the secretory protein flow ogy, the vast majority of vps mutants (>75%) contain a normal (Graham and Emr, 1991) and delivered to the vacuole via a or near-normal vacuole structure (classes A, D, E and F). This prevacuolar endosome intermediate (Vida et al., 1993). In suggests that these mutants are competent to construct and order to identify the cellular machinery involved in Golgi-to- maintain a vacuole structure, yet have specific protein targeting vacuole protein sorting and delivery events, several mutant and sorting defects. Some of these defects may involve the dis- selection schemes have been designed that detect mislocaliza- ruption of specific vesicular trafficking events that are respon- tion of soluble vacuolar proenzymes (Bankaitis et al., 1986; sible for the delivery of distinct subsets of vacuolar proteins Rothman and Stevens, 1986; Robinson et al., 1988; Rothman from a late Golgi compartment to the vacuole. et al., 1989). Using these selection schemes, a large number of Analysis of protein movement through various stages of mutants have been identified that missort and secrete vacuolar both the secretory and vacuolar protein localization pathways proteins. Together, these vacuolar protein sorting (vps) mutants has uncovered a number of protein families that direct define greater than 40 complementation groups that have been common events (i.e., transport vesicle targeting and fusion) divided into six distinct morphological classes (Banta et al., at each stage of the transport process. One such family 1988; Raymond et al., 1992). Class A vps mutants display includes the yeast rab proteins Ypt1p, Vps21p and Sec4p. wild-type morphology: one to three large vacuole structures are These small GTP-binding proteins appear to be involved in present in each cell. The vacuoles of class B vps mutants are the targeting and/or fusion of vesicular transport intermedi- 3450 C. R. Cowles, S. D. Emr and B. F. Horazdovsky ates at distinct stages of the transport pathway. Ypt1p is Plasmid constructions involved in ER-to-Golgi vesicle movement (Goud et al., Recombinant DNA manipulations employed in the construction of 1988). Vps21p functions in the vacuolar protein sorting plasmids were performed as described previously (Maniatis et al., pathway (Golgi to vacuole; Horazdovsky et al., 1994), and 1989), with the exception of DNA fragment isolations carried out by Sec4p is required for delivery of secretory vesicles from the the glass bead method of Vogelstein and Gillespie (1979). The CEN- Golgi to the plasma membrane (Bruno et al., 1988). Another based plasmid pVPS45-10 was generated by inserting the SmaI-PvuII group of proteins, the Sec1 family, also appears to function fragment of library plasmid pVPS45-1 (containing VPS45, refer to in vesicle targeting or fusion. In yeast, the Sly1, Sec1 and Fig. 1A) into the SmaI site of pRS414 (Sikorski and Heiter, 1989). The same SmaI-PvuII fragment was also inserted into the SmaI site Vps33 (Slp1) proteins are required for protein delivery from of pRS424 (Sikorski and Heiter, 1989) to create the 2µ-based plasmid ER to Golgi, Golgi to plasma membrane, and Golgi to pVPS45-15. For sequence analysis, plasmid pVPS45-9 was con- vacuole, respectively (Novick et al., 1980; Banta et al., 1990; structed by inserting the ClaI-SacI fragment of pVPS45-10 (contain- Wada et al., 1990; Dascher et al., 1991). Distinct Sec1p ing the SmaI-PvuII fragment of pVPS45-1) into the ClaI and SacI sites family members also have been implicated to function in the of pBluescript KS (Stratagene). Generation of pVPS45-6 was vesicular trafficking events of other eukaryotes. In achieved by ligating the EcoRV-EcoRV fragment of pVPS45-1 into mammalian neuronal cells, n-Sec1 has been shown to interact the SmaI site of pBluescript KS. Plasmid pVPS45-5 was constructed with the plasma membrane protein syntaxin (Hata et al., 1993; by inserting the BglII-BglII segment of pVPS45-1 into the BamHI site Garcia et al., 1994; Pevsner et al., 1994). Syntaxin is thought of pBluescript KS. pVPS45-21 consisted of the EcoRV-HindIII to serve as one of the receptor molecules involved in the segment of pVPS45-1 inserted at the SmaI and HindIII sites of pBlue- docking of synaptic vesicles with the plasmalemma (Söllner script KS. Plasmids pVPS45-4 and pVPS45-7 were generated by introducing the BglII-PvuII or SmaI-BglII fragments of pVPS45-1, et al., 1993a,b). UNC18 has been proposed to serve a similar respectively, into the SmaI and BamHI sites of pRS414. The integra- function in Caenorhabditis elegans (Hosono et al., 1992; tive mapping plasmid, pVPS45-17, was constructed by inserting the Pelham, 1993). Though members of the Sec1 protein family ClaI-SacI fragment of pVPS45-10 into the ClaI and SacI sites of and the rab-like GTP-binding proteins clearly play critical pRS304 (Sikorski and Heiter, 1989). roles in vesicle-mediated protein trafficking, their exact An intermediate plasmid construction was performed in generating functions remain unclear. the vps45 deletion/disruption. Plasmid pVPS45-9 was digested with Here we report the characterization of one of the VPS gene EcoRV and BglII, removing a large portion of VPS45 (see Fig. 1A), products involved in delivery of proteins to the yeast vacuole. and the remainder of the vector sequences were isolated and purified. VPS45 encodes a 67 kDa homolog of Sec1p. Subcellular local- pHIS3 (from E. Phiziaky) was digested by XhoI, blunted by a Klenow ization of Vps45p suggests that it is peripherally associated fill-in reaction, and digested with BamHI. The resulting XhoI(blunt)- with cellular membranes (potentially including Golgi and BamHI fragment containing the HIS3 gene was isolated and ligated into the EcoRV and BglII sites of pVPS45-9 (lacking the VPS45 endosomal membranes, as well as membrane vesicles). vps45 coding sequences) to generate disruption plasmid pVPS45-19. In mutants missort multiple vacuolar hydrolases, are temperature order to construct a TrpE-Vps45 fusion protein, plasmid vector sensitive for growth, and exhibit a class D vacuole morphol- pATH2 (containing the trpE coding sequences; Dieckmann and ogy. Interestingly, vps45 deletion mutants accumulate what Tzagoloff, 1985) was digested with SmaI and HindIII. Plasmid appear to represent aggregates of intermediate transport pVPS45-9 was digested with EcoRV and HindIII, and

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