VPS21 encodes a rab5-like GTP binding protein that is

Transcription

1 The EMBO Journal vol.13 no.6 pp , 1994 VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins Bruce F.Horazdovsky1, Gerald R.Buschl and Scott D.Emr2 Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, C , US 2Corresponding author 'The first two authors contributed equally to this study Communicated by D.Meyer Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps2l, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps2lp is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and >70% sinilarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps2l/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps2lp function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 C, (ii) missort multiple vacuolar proteins, (iii) accumulate nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kda protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps2lp. We propose that Vps2lp functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole. ey words: GTP binding protein/sorting/vacuole/vps21/ yeast Introduction The compartmentalized nature of eukaryotic cells requires a specific and highly efficient means of transporting proteins to and from various intracellular locations. In most cases, this involves vesicle-mediated transport systems that deliver their specific set of soluble and membrane protein constituents to a defined subcellular location. One of the best characterized vesicle-mediated transport systems is found in the secretory pathway (reviewed in Pryer et al., 1992). Secreted proteins transit between a number of distinct intracellular organelles: transport from the ER to the Golgi complex, movement to and from distinct Golgi cisternae and finally delivery from the Golgi to the cell surface. Each of these transport steps appears to depend on vesicular intermediates to facilitate the protein delivery reactions (Palade, 1975; Pfeffer and Rothman, 1987; Holcomb et al., 1988). Due to the directionality of the secretory pathway and the large number of different compartments involved, the targeting and fusion of these vesicle intermediates must be highly regulated. In vitro reconstitution assays as well as the use of genetic approaches in yeast have uncovered a large number of soluble and membrane-associated components required for the generation, targeting and fusion of transport vesicles as they move through the secretory pathway (Pryer et al., 1992). One group of proteins required in this process are members of the ras-like small GTP binding protein superfamily, the rab proteins (Goud et al., 1988; Pfeffer, 1992; Schwaninger et al., 1992). Distinct members of this family regulate specific steps in the secretion pathway by promoting the targeting and/or fusion of vesicular intermediates with the appropriate membrane targets in a GTP-dependent manner (Pryer et al., 1992; Walworth et al., 1992). This level of specificity is essential to maintain the unique characteristics of the secretory pathway organelles. In Saccharomyces cerevisiae, the delivery of proteins to the lysosome-like vacuole involves movement of proteins to and through the early stages of the yeast secretory pathway (Stevens et al., 1982). In a late Golgi compartment, soluble vacuolar proteins are sorted away from bulk secretory protein flow and are specifically targeted to the vacuole (Graham and Emr, 1991). Similar to the targeting of soluble lysosomal proteins in mammalian cells (reviewed in ornfeld, 1992; ornfeld and Mellman, 1989), vacuolar proteins appear to be delivered from the Golgi to the vacuole via an endosomal intermediate (Vida et al., 1993). Subcellular fractionation analysis of the vacuolar protein delivery reaction indicates that carboxypeptidase Y (CPY) transits through a prevacuolar endosomal compartment before delivery to the vacuole (Vida et al., 1993). The movement of vacuolar proteins from the Golgi to the endosome and from the endosome to the vacuole is likely to involve vesicular transport intermediates as well as the activity of specific small GTP binding proteins. In the yeast secretory pathway, the small GTP binding proteins Yptlp and Sec4p are required for vesicle transport steps between the ER and Golgi and the Golgi and plasma membrane, respectively (Goud et al., 1988; Becker et al., 1991). Mutations in these genes result in a defect in protein secretion and the accumulation of vesicle intermediates (Goud et al., 1988; Segev et al., 1988). Similarly, mutations in the genes encoding small GTP binding proteins involved in the delivery of proteins from the Golgi to the vacuole would be expected to lead to a vacuolar protein sorting defect and possibly the accumulation of vesicle carriers. Several genetic selections have been used to identify a large group of mutants that missort a variety of vacuolar proteins including CPY, proteinase (Pr) and proteinase B (PrB) Oxford University Press 1 297

2 B.F.Horazdovsky, G.R.Busch and S.D.Emr (Bankaitis et al., 1986; Rothman and Stevens, 1986; Robinson et al., 1988; Rothman et al., 1989). Unlike wild type cells, vacuolar protein sorting (vps) mutants missort and secrete these vacuolar proteins as their Golgi-modified precursors. Together, the vps mutants define > 40 complementation groups, presumably reflecting the complex nature of this sorting reaction (lionsky et al., 1990; Raymond et al., 1992). Some vps mutants lack a normal vacuole compartment which indicates that they are defective in the delivery of both soluble and membrane constituents of the vacuole (Banta et al., 1988). Somewhat surprisingly, most of the vps mutants still contain near-normal vacuoles, indicating that many of the vps mutants do not eliminate membrane and protein traffic to the vacuole but rather affect very specific protein recognition, sorting or targeting reactions. In order to gain insight into the trans-acting cellular machinery involved in vacuolar protein sorting, we characterized the gene and gene product affected in vps2l mutants. vps2l mutant cells contain a vacuole but exhibit a severe defect in the sorting of the soluble vacuolar hydrolases CPY and Pr (Robinson et al., 1988). Here we show that VPS21 encodes a 22 kda protein that exhibits striking homology to the rab family of small GTP binding proteins. The highest similarity is to rab5 which has been shown to function in the early stages of the mammalian endocytic pathway (Chavrier et al., 1990; Gorvel et al., 1991; Bucci et al., 1992). In yeast, however, we find that Vps2 Ip is required for the sorting of vacuolar proteins but not for the internalization of the endocytosed mating pheromone, ae-factor. Together our data indicate that the VPS21 gene product is required for the targeting and/or fusion of vesicular carriers that function in the transport of vacuolar proteins between the Golgi and the endosome or the endosome and the vacuole. Results Characterization and disruption of the VPS21 locus Originally, many of the vacuolar protein sorting mutants were isolated using a CPY-invertase gene fusion-based selection scheme (Bankaitis et al., 1986; Robinson et al., 1988). In wild type cells, the CPY-invertase fusion protein is quantitatively delivered to the vacuole due to the localization information contained in the CPY portion of the hybrid (Johnson et al., 1987). However, in vps mutants the CPY - invertase fusion protein is mislocalized and secreted to the cell surface. This results in a selectable phenotype, the ability of these mutants to grow on sucrose (an invertase substrate). We exploited the CPY - invertase missorting phenotype to clone the wild type VPS21 gene. vps2l-2 mutant cells (SEY2 1-2) expressing a CPY -invertase fusion protein were transformed with a yeast genomic library (CEN, LEU2). One hundred and twenty thousand Leu+ transformants were isolated and subsequently screened for the proper localization of the CPY-invertase hybrid by a simple colorimetric plate assay designed to detect extracellular invertase activity (Paravicini et al., 1992). Transformants carrying a VPS21 complementing plasmid restored the vacuolar localization of the CPY-invertase fusion protein and gave rise to white colonies. Transformants carrying noncomplementing plasmids produced red colonies as a result of the mislocalization of the CPY-invertase fusion to the cell surface. This method yielded three 1298 complementing clones, all of which shared common genomic fragments. Plasmids were recovered from the transformants and then reintroduced into SEY21-2 as well as other vps2l mutant strains. In each case, the plasmids complemented the CPY - invertase missorting phenotype. One of the complementing library plasmids contained a 7.2 kb genomic DN insert (Figure I). The complementing activity was further mapped to a 1.2 kb ClaI-BglII fragment. The complementing genomic fragment was subsequently shown to correspond to the VPS21 locus by integrative mapping experiments (see Materials and methods). The complementing ClaI -BglII fragment was subcloned into the multicopy 2, vector prs424 to generate pbhy Transformation of strain SEY21-2 (vps2l-2) and wild type cells with pbhy resulted in the >20-fold overproduction of Vps2ip (Figure 5, see below). These transformants also exhibited a slight Vps- phenotype, indicating that overexpression of Vps2lp may lead to a partial dominant-negative phenotype. - C B (' I CC SE BPv E H P SpH. a I. I*,.. I. I I I ai I S CWT WT 25C c i's2 1 L p-b Sp 1 J.7.. If b Plt B p. I. vps2 1 VPS21 vps2 I V'PS21 Fig. 1. Characterization and disruption of the VPS21 locus. () restriction map of the 7.2 kb genomic DN contained in the VPS21 complementing plasmid clone, pgby21-1. The VPS21 coding sequence is indicated by a black arrow; C, ClaI; S, Sail; E, EcoRV; B, Bglll; Pv, PvuII; H, HindmI; P, PstI and Sp, SphI. (B) The 0.7 kb Sall -Bglll fragment containing the VPS21 coding sequence was replaced with a HIS3 gene cassette to generate the deletion/disruption strain GBYlO. (C) The growth characteristics of wild type cells (SEY6210), cells carrying the vps2l null mutation (vps2l, GBYIO) or vps2j cells carrying the complementing plasmid pgby21-5 (VPS21 CEN) on YPD plates are shown following 3 days of incubation at either 25 or 38 C

3 GTP binding protein required for protein sorting To determine the phenotypic consequences of deleting the VPS21 gene, the VPS21 coding sequence was replaced with a DN fragment containing the yeast HIS3 gene (Figure IB). parental diploid strain, BHY10.5 (VPS211VPS21, his3-2001his3-200, leu2-3,112 CPY-invertase::LEU2/ leu2-3,112 CPY -invertase::leu2), was transformed with a ClaI-PvuH fragment containing the deletion of VPS21. His+ transformants were isolated and the diploid transformants were sporulated. s expected, a segregation pattern of 2:2 Vps- (CPY-invertase secreted) His+:Vps+ (CPYinvertase not secreted) His- was observed. ll haploid progeny of the 10 tetrads analyzed were viable. Disruption of the VPS21 locus was confirmed using PCR amplification of genomic DN isolated from representative haploid segregants (data not shown; see Materials and methods) (Herman and Emr, 1990). The viability of vps2j cells indicated that VPS21, like other characterized VPS genes, is not essential for growth. The vps2j cells did, however, exhibit significantly slower growth at 38 C than either the wild type controls or the complemented vps2l mutants (Figure 1C). The effect of deleting VPS21 on native vacuolar protein sorting and vacuole morphology was also analyzed. Vacuole morphology was examined using the vacuole-specific vital stain 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFD) (Pringle et al., 1989). Consistent with previously published observations using vps2l mutants (Banta et al., 1988), vps2l cells showed only a slightly enlarged vacuole when compared with wild type cells. Substantially fewer vacuole segregation structures (which are involved in vacuole inheritance from mother to daughter cells) were observed in vps2] cells (data not shown). These results indicate that the VPS21 gene product is not required to maintain vacuole structure but may play some role in vacuole inheritance. In addition, the morphology of wild type and vps2j mutant cells was examined by electron microscopy. s seen in Figure 2, vps2j cells contain a vacuole but also accumulate nm vesicles (small arrows). Quantitative analysis revealed that these vesicles - were present in vps2j cells at 3-fold higher levels than in wild type cells (based on a comparison of the number of vesicles/cm2 in > 100 cell sections for both mutant and wild type cells). Each vesicular structure was surrounded by a membrane bilayer (Figure 2, large magnification). These small vesicles were distinct from the nm secretory vesicles that accumulate in sec mutants that block Golgi to cell surface traffic (Novick et al., 1980). Such secretory vesicles can be seen in Figure 2 (large arrowhead) clustering at the site of a newly forming bud. Vacuolar hydrolases undergo a number of compartmentspecific modifications as they transit through the early stages of the yeast secretory pathway (Stevens et al., 1982). These modifications result in easily distinguishable precursor forms of vacuolar proteins. The ability of wild type, vps2j cells and vps2j cells carrying the complementing plasmid pgby21-5 (VPS21, CEN) to sort native vacuolar proteins was examined by a pulse -chase analysis of the soluble vacuolar hydrolases, CPY and Pr, as well as the vacuolar integral membrane protein alkaline phosphatase (LP). In the experiment shown in Figure 3, whole cells were labeled for 10 min and chased for 30 min after the addition of excess unlabeled methionine and cysteine. The cells were then converted to spheroplasts, separated into intracellular (I) and extracellular (E) fractions and CPY, Pr or LP were recovered by immunoprecipitation. Wild type cells and vps2j cells transformed with pgby21-5 properly delivered X4' N V N *ek _.1?.f.\ vp s?21 Ns, /- n... IN \ i, i- CS w f jor-- i op,- 4..t s Fig. 2. Morphological analysis of vps2l mutant cells. Wild type cells (WT, SEY6210; top panel) or cells carrying the vps2l disruption (vps2j, GBYIO, center panel) were grown to mid-log phase, fixed and prepared for electron microscopic analysis. In the upper two panels, N marks the nucleus; V, the vacuole; M, the mitochondria. The bars represent 0.5 m. The small arrows in the center panel highlight the nm vesicles; the large arrowhead highlights nm secretion vesicles. The dashed boxed area in the center panel is shown enlarged in the bottom panel. The bar in the bottom panel represents 0.1 gm. 1299

4 *' t B.F.Horazdovsky, G.R.Busch and S.D.Emr ji_ Z ;;....,r.,,,. _. f * q; ito i,, ;\ t,1,gf',2! _.--1..: -4::.. ::,";m -:154 o, i,::i:.. Fig. 3. Intracellular sorting of vacuolar hydrolases. Yeast cells were labeled for 10 min at 30 C with Tran35S-label, chased for 30 min at 30 C, then spheroplasted. The labeled cultures were separated into pellet (internal, I) and supernatant (external, E) fractions. The presence of CPY, Pr and LP in these fractions was determined by immunoprecipitation. The yeast strains used in this analysis included SEY6210 (WT, lanes 1 and 2), GBYIO (vps2l, lanes 3 and 4), and GBYIO carrying the complementing plasmid pgby21-5 (vps211vps21 CEN, lanes 5 and 6). The migration positions of Golgi-modified precursors (p2 and pro) and mature (m) proteins are shown. CPY, Pr and LP to the vacuole as evidenced by the presence of these proteins in the I cell fraction as their mature vacuolar forms (mcpy, mpr and mlp) (Figure 3, lanes 1 and 5). In contrast, the vast majority of CPY, Pr and LP were found as their Golgi-modified precursor forms (p2cpy, propr, prolp) in vps2j cells. Greater than 80% of the p2cpy was secreted into the E fraction from these cells. ProPr was also secreted from vps2j cells, although a significant portion was also retained inside the cells (Figure 3, lanes 3 and 4). Not surprisingly, LP, the integral vacuolar membrane protein, was also retained in the vps2j cells (Figure 3, lane 3). The presence of a small amount of intracellular mature CPY, Pr and LP indicated that a fraction of these soluble and membrane hydrolases was reaching the vacuole or a vacuole-like compartment. However, much of the CPY and Pr that was retained within the mutant cells was present as aberrantly processed forms which migrate between the pro and mature forms (Figure 3, lane 3). VPS21 sequence analysis The nucleotide sequence of VPS21 was determined by sequencing a set of nested exonuclease HI-generated deletion constructs that spanned the minimum complementing VPS21 DN fragment. single large open reading frame of 210 if amino acids was identified capable of coding a protein with a molecular mass of Da (Figure 4). This small protein lacked hydrophobic sequences that might correspond to a signal sequence or transmembrane domain. When the amino acid sequence was compared with other known protein sequences, a striking homology was noted between Vps2 ip and many ras-like GTP binding proteins. The highest similarity was found with members of the rab GTP binding protein family, especially the rab5 proteins of other eukaryotic cells (Figure 4B) (Chavrier et al., 1990; nuntalabhochai et al., 1991; rmstrong et al., 1993a). This homology was found to be -50% identity and -70% similarity over the whole length of the various rab5 proteins, with the most highly conserved regions clustered within the proposed GTP binding motifs and the effector domain of these proteins. Mammalian rab5 protein has been shown to be involved in the trafficking of endocytic vesicles through the early stages of the endocytic pathway (Chavrier et al., 1990; Gorvel et al., 1991; Bucci et al., 1992). Further inspection of the Vps2 Ip sequence revealed the presence of two C-terminal cysteine residues (CXC motif) that, in the case of many ras-like GTP binding proteins, have been shown to be the site of modification by an isoprenyl group (Glomset et al., 1990; Powers, 1991; Magee and Newman, 1992). This modification is thought to be responsible for the membrane association of many GTP binding proteins (see below). Identification of the VPS21 gene product TrpE-Vps2l fusion protein was used to generate polyclonal antiserum that would recognize Vps2lp. s shown in Figure 5, this antiserum immunoprecipitated two Vps21 protein species in wild type cells (23 and 22 kda, lanes 2 and 3) that were absent in cells that carried the vps2l deletion mutant (lane 1). In addition, when immunoprecipitation experiments were performed with preimmune serum, no protein bands were detected (data not shown). Vps21p was synthesized initially as a 23 kda protein which is in agreement with the molecular mass predicted from sequence analysis (lane 2). Following a 60 min chase period, the 23 kda Vps2 ip species appeared to be converted into the smaller 22 kda species (lane 3). When the VPS21 coding sequence was placed in a multicopy plasmid, >20-fold overproduction of Vps2 Ip was noted compared with wild type cells (lanes 4 and 5). s seen in the wild type cells, the overexpressed Vps2lp was initially present as the larger 23 kda form that was partially converted to the smaller 22 kda form during the 60 min chase period. The vast majority of the overexpressed material remained as the larger 23 kda species, suggesting that overexpressing Vps2ip may have saturated a component(s) involved in a processing or modification event (see below). In both wild type and overexpressing cells, Vps2lp was fairly stable; after a 60 min chase period, no significant loss of Vps21p was noted (Figure 5, lanes 3 and 5). When compared with the expression level of the well-characterized vacuolar hydrolase - CPY (representing 0.1 % total yeast cell protein), Vps2 Ip is found to be present in wild type cells at - levels of % of total yeast protein. These results indicate that the VPS21 gene codes for a relatively abundant and stable protein with a 'mature' molecular mass of 22 kda. 1300

5 , GTP binding protein required for protein sorting I N L N E G F B I V G S 1 G F L F I W C T C Q E R F 73 3 L V V Y ID V T P Q 97 L H E Q S D I I I L QB G G V E E G B L 2.45 F E T S T C B N V N D V F L 169 Fl T E E Q N S S N B R B S N N.1 93 V D G T S N S c S C * 212.' _ B S.cL-I)ps2 1 p Canine rah5 S.P. YPt5P Plant rab; Motif 1I WTGE I * * ~H L 9 RE IGEL M NT SV TS M N P G T PI) NGC P N T G N C aof M Si N T P N V V T N 0 M S SG N IN ED~ E ElI Q3 9 - Motit, IN' I *L ' Q C. F I GTIP BSinding Miotif I G x xx xg E U N& E N G H EF 0 L ~D D DY R.D V EFO0 II I Effector Domiaini I Gx x RVT I NE H ~V C L DDT~ I~~~*LP I DE N F S L V N D Miotif III N E E R N R SSPN S.. N NQ0 E R~ sgn P NMfMk L D D GE DV LG GEP TEE NSSNERESNNORDDD NNDGDSNIMBSC 211 S M ElI M PNEFNENP NPGN.N.S R G RG D T EP T QPDR SQC C SN 21I5 E E L T L P E D LNE.I.RG V N RG N S E R P OPGEGoSc :`1 T D I Y E L P V P E N P Ta0....M V P N G P G Ea V SDSE C 2IH R Fig. 4. The nucleotide sequence of VPS2J and amino acid comparison with rab5 protein homologues. () The nucleotide sequence and the deduced amino acid sequence of VPS2J are shown. The sequence accession number for VPS2J is Z (B) Comparison of Vps2lp (S.c Vps2lp) with rab5 homologues including canine rab5 (Chavrier et al., 1990), Ypt5p of Schizosaccharomyces pombe (S.p. Ypt5p) (rmnstrong et al., 1993a) and RHI rabidopsis thaliana (Plant rab5) (nuntalabhochai et al., 1991). Regions of amino acid identity are shown in black boxes. The GTP binding motifs and the effector domain corresponding to similar domains in ras/rab proteins are denoted by the large boxes. Vps2 ip associates with multiple membrane compartments Subcellular fractionation was carried out to identify the intracellular location of Vps2lIp. Wild type spheroplasts were labeled with Tran35S label, chased and lysed under conditions that maintain the integrity of intracellular organelles (Horazdovsky and Emr, 1993). The cell lysate was subjected to centrifugation at 500 g to remove unlysed spheroplasts. The cleared lysate was then centrifuged at g to generate supernatant (S 13) and pellet (P 13) fractions. The 13 fraction was then spun at g to generate a second set of supernatant (S 100) and pellet (P1 00) fractions. The relative levels of Vps2lIp and organelle marker proteins were determined by immunoprecipitation. s shown in Figure 6., most of Vps2lIp was found in a particulate cell fraction (lanes and 3). Greater than 75% of the total Vps2 p present in wild type cells was found in the P13 and P100 subcellular fractions. In some experiments, > 90% of Vps2 Ilp was found in these fractions. The majority of the sedimentable Vps2l1p was recovered in the lower speed pellet (P13). This portion of Vps2lIp showed a fractionation profile similar to the vacuolar membrane marker LP (lionsky and Emr, 1989) and clearly fractionated away from a Golgi marker protein, ex2p (Redding et al., 1991) (Figure 6). However, other membrane marker proteins are also found in the P13 fraction, including the ER protein HMG Co reductase (Wright et al., 1988) and the plasma membrane TPase (Serrano, 1993) (data not shown). nother portion of the sedimentable Vps2 ip was recovered in a distinct particulate cell fraction, the g pellet (P100). ex2p (the processing endopeptidase required for the maturation of mating pheromone et-factor) also was recovered in this higher speed cell pellet fraction. Previous fractionation studies have identified small membrane vesicles as components of the P100 cell pellet as well (Walworth et al., 1989; Bucci et al., 1992). Thus, Vps2lp can be proposed to associate with a number of intracellular membranes. To determine the nature of the association of Vps2 lp with 1301

6 B.F.Horazdovsky, G.R.Busch and S.D.Emr Strain: vps2l WT 2g-VPS2I Chase: 6I F1 Y P13 SIOO PI00-22 kl),iii::..4. VPS2_ I p - X. - "*.::.111. I;.;. 23 D -naimwy- -22 k. D -Vac Fig. 5. Identification of the VPS21 gene product. Wild type cells (WT; SEY6210, lanes 2 and 3), cells carrying a vps2l null mutation (vps2l; GBYIO, lane 1) or wild type cells carrying the 2 / plasmid pbhy21-28 (2,u-VPS2J; lanes 4 and 5) were labeled for 10 min with Tran35S label at 300C. The labeling was either terminated by the addition of trichloroacetic acid (to 5%) (lanes 1, 2 and 4), or a 60 min chase period at 300C was included prior to trichloroacetic acid precipitation (lanes 3 and 5). Protein pellets were suspended and immunoprecipitated with Vps2lp-specific antibody and the antigen-antibody complexes were subjected to SDS-PGE and fluorography. Molecular mass is shown to the right in kda and was determined by a comparison of Vps2lp migration with stained protein standards. Exposure time for the panel including lanes 4 and 5 was one-tenth of that for the panel including lanes 1-3. the P13 and P100 particulate cell fractions, cleared cell lysates generated from wild type spheroplasts were pretreated with various reagents prior to centrifugation at g (Figure 7). Pretreatment of the particulate cell fraction with 1 M NaCl had litfle or no effect on the partitioning of Vps2lp with the P100 pellet (Figure 7, lanes 5 and 6). This result suggested that Vps2 lp is not associated with the particulate cell fraction via ionic interactions with another protein(s). In contrast, when the particulate cell fraction was treated with a detergent solution (1% Triton X-100), Vps2 Ip was quantitatively extracted into the S100 fraction (Figure 7, lanes 3 and 4). This result indicated that Vps2lp is associated with intracellular membranes. In addition, when the P13 and P100 cell fractions were subjected to sucrose density gradient analysis, portions of Vps2lp were found to colocalize with the vacuolar membranes, Golgi membranes and light membrane fractions of unknown composition, but fractionated away from the dense plasma membrane (data not shown). Modification of Vps21p is required for its association with cellular membranes Many small GTP binding proteins are associated with cellular membranes through prenyl modification of a C-terminal cysteine (Glomset et al., 1990; Powers, 1991; Magee and Newman, 1992). In the case of several rab proteins, the prenyl modification is a geranylgeranyl moiety. To determine whether Vps21p is associated with cellular membranes through this type of modification, the subcellular location G6PDIH-- WRn~0'a Golcgi -Cyto) Fig. 6. Subcellular fractionation of the VPS21 gene product. Wild type cells were converted to spheroplasts, labeled with Tran35S-label for 30 min and chased 30 min at 30 C, lysed and subjected to sequential differential centrifugation. Equivalent amount of the g pellet fraction (P13, lane 1), as well the g supernatant (Sloo, lane 2), and the g pellet fraction (Ploo, lane 3) were subjected to quantitative immunoprecipitations with antiserum directed against Vps2lp, the vacuole membrane protein LP, the Golgi membrane protein ex2p and the cytosolic protein G6PDH..cx2p-...- Vps2Ip- No 1C. IM Wash 1Triton NaCI Is P 11S P! S P Fig. 7. The association of Vps2lp with the particulate cell fraction. Wild type cells were lysed as described in Figure 6. Cleared cell lysate (supematant of a 500 g centrifugation) was pretreated with buffer alone (lanes 1 and 2), 1% Triton X-100 (lanes 3 and 4) or 1 M NaCl (lanes 5 and 6) prior to centrifugation at g. The presence of Vps2lp in the resultant supematant (S) and pellet (P) fractions was determined by quantitative immunoprecipitation. of Vps2lp was examined in a strain carrying a bet2 mutation (Rossi et al., 1991). BET2 has been shown to encode a homolog of the mammalian geranylgeranyl transferase a

7 'ps21pi I_ X Fig. 8. The dependence of the membrane association of Vps2lp on post-translational modification. Wild type cells (WT; SFNY26-6, lanes 1 and 2), bet2 mutant cells (bet2; NY1 19, lanes 3 and 4), cells expressing the C208,210S Vps2l mutant protein (C-S; GBY10/pBHY21-15, lanes 5 and 6) or wild type cells overexpressing Vps2lp (24 VPS21; SEY6210/pBHY21-28, lanes 7 and 8) were converted to spheroplasts, 35S-labeled, chased and lysed. The cell lysates were cleared (500 g centrifugation for 10 min) and then centrifuged at g for 60 min. The presence of Vps2lp in the supernatant (S) or pellet (P) fractions was determined by immunoprecipitation. In the case of WT and bet2 cells, spheroplasts were incubated at 37 C for 15 min prior to a 10 min label and 30 min chase at 37 C. C- S cells and 24 VPS21 cells were labeled for 30 min and chased for 30 min at 30 C. subunit (Rossi et al., 1991; rmstrong et al., 1993b; Jiang et al., 1993). If geranylgeranylation is required for Vps2 Ip membrane association, then Vps2 Ip should be found in a soluble fraction in a bet2 temperature-sensitive mutant strain incubated at the nonpermissive temperature (Rossi et al., 1991). Spheroplasts were shifted to 37 C (inactivating Bet2p), then labeled, chased, lysed and cleared of unbroken spheroplasts by low speed centrifugation. The cleared lysate was then spun at g and the location of Vps2 Ip was determined by immunoprecipitation of the supernatant (Sl 00) and pellet (P100) fractions. s shown in Figure 8, Vps21p was found in the S 100 fraction of bet2 spheroplasts (Figure 8, lanes 3 and 4). The BET2 parental strain properly localized Vps21p to the membrane fraction (Figure 8, lanes 1 and 2). This strongly indicated that a geranylgeranyl modification is required for the association of Vps21p with both the P13 and P100 subcellular membrane fractions. Interestingly, Vps21p migrated with an apparent molecular mass of 23 kda in the bet2 mutant cell extracts (Figure 8, lane 3), similar to that seen after pulse labeling Vps21p during a pulse-chase experiment (Figure 5). These observations indicated that the geranylgeranyl modification of Vps2 lp accounted for the 1 kda mobility shift of Vps2 Ip at later chase points. This shift may be the result of the geranylgeranyl modification itself; alternatively, prenylation may be required for a secondary modification/processing event to take place which in turn results in the mobility change. s previously mentioned, the C-terminal cysteines of rab proteins are the site of geranylgeranyl modification (Magee and Newman, 1992). To determine whether this was the case for Vps2 Ip, subcellular fractionation was carried out in a vps2l mutant strain in which both of the C-terminal cysteine residues of Vps2 lp had been changed to serine (C208,2 OS) (see below). Cells carrying this mutant form of VPS21 expressed a stable protein that no longer associated with cellular membranes but was instead recovered in the S100 soluble fraction (Figure 8, lanes 5 and 6). Like that in the bet2 mutant cells, the soluble Vps21p was also present in the larger 23 kda form, further supporting the notion that geranylgeranylation is responsible for the 1 kda mobility GTP Bin~dinlg Nlotit GEVGS N,I S1 GTP binding protein required for protein sorting WIF sbcf2 C - -s 2u IPS'/ I S i' 11 S P.lS li) 1 S- ) -.ffmot - 0 Efftector G;-G *_llimig_ 40_ --' _- k 1) 1)omain rzrlil'itt Site 4IM -W-'kI) TIGF CSC css S. j, S N p2cpy-. riiic.. P N - B _ T, 9 C,)~l S WT./i I() S W\T Vps2 I p la._ qmm C VpsIp '11, i....'' 40b: j 4 5 -i k,) -22 kd -23 kid -22 kd) Fig. 9. Mutational analysis of Vps2lp. () GBYIO cells (vps2j) carrying plasmid pbhy21-11 (S21-N vps2l point mutant, lane 1), plasmid pbhy21-12 (T39- vps2l point mutant, lane 2), pbhy21-15 (C208,210-S vps2l point mutant, lane 3), or pgby21-2 (VPS21) and GBYIO cells (vps2j) were labeled for 10 min with Tran35S-label and chased for 30 min at 30 C. Cells were then lysed and CPY immunoprecipitated. The positions of Golgi-modified p2cpy (69 kda) and vacuolar mature CPY (61 kda) are indicated. (B) Unlabeled cell extracts from log phase cultures of the strains listed above were prepared and immunoprecipitated with Vps2ip antiserum. The antigen-antibody complexes were resolved using SDS-PGE and subjected to Western analysis using Vps2lp antiserum. (C) Duplicate samples of the Vps2lp unlabeled immunoprecipitations were resolved on an SDS-polyacrylamide gel, renatured and transferred to nitrocellulose. The blot was incubated in the presence of [a-32p]gtp, and the [a-32p]gtp subsequently bound to Vps2lp was visualized by autoradiography. shift seen in the pulse -chase experiments described earlier. The bulk of overexpressed Vps2lp also fractionates as a soluble protein. In a strain that overproduced Vps2lp at levels >20-fold over wild type, >90% of the overexpressed material was found in the 5100 as the 23 kda ('nonprenylated') form (Figure 8, lanes 7 and 8). This result indicated that the overexpressed Vps2lp was not prenylated possibly because either the geranylgeranyl transferase or the cellular pool of the transferase substrate (geranylgeranyl-pp) (Glomset et al., 1990) was limiting. When equivalent gel loadings were used to compare the subcellular fractionation of Vps2 Ip in wild type and overexpressing cells, the absolute amounts of the 22 kda membrane-associated form of Vps2 lp were similar in wild type and Vps2lp overexpressing cells. 1303

8 B.F.Horazdovsky, G.R.Busch and S.D.Emr Mutational analysis of VPS21 To examine further the role of the GTP binding domains, the effector domain and the prenylation site found within Vps2 Ip, site-directed mutagenesis was carried out to change amino acids within these conserved segments of the protein (Deng and Nickoloff, 1992). Three mutants were generated and analyzed. In the first mutant, the conserved serine residue at position 21 in GTP binding motif I was changed to asparagine (S2 IN) (Figures 9 and 4). Similar changes in other GTP binding proteins are thought to stabilize the GDPbound form of the protein and lead to a dominant-negative phenotype (Freig and Cooper, 1988; Tisdale et al., 1992). In the second mutant, a conserved threonine in the effector domain of Vps2lp was changed to alanine (T39, Thr39 to la). The corresponding mutation in ras results in a defect in transforming ability and reduced levels of GP-stimulated GTPase activity (Cales et al., 1988). Finally, as described above, in the third mutant the C-terminal cysteine residues were changed to serines (C208,2 OS). ll of the rab proteins described to date contain C-terminal cysteine residues and these cysteine residues have been shown for many to be the site of geranylgeranylation (Magee and Newman, 1992). Mutating GTP binding motif I, the effector domain or the C-terminal cysteines of Vps2 lp resulted in a severe vacuolar protein sorting defect. In pulse-chase experiments, nearly all of the CPY accumulated as the Golgi-modified p2 precursor form in the mutant strains (Figure 9, lanes 1-3). Similarly, in the vps2l mutant, very little mature CPY could be detected (Figure 9, lane 5). One possibility is that these point mutations destabilize the Vps21 protein and therefore lead to a CPY sorting defect. To test this, Western analysis was performed on cell extracts from strains expressing the mutant proteins. s shown in Figure 9, the mutant Vps2l proteins were present at a level similar to that of the wild type protein (panel B, lanes 1-4). However, unlike wild type Vps21p, most of the mutant Vps2l proteins migrated as the 'nonprenylated' 23 kda form. This was expected for the C-terminal cysteine mutant (lane 3), but was not expected for the S2 IN GTP binding motif mutant or the T39 effector domain mutant (lanes 1 and 2). Only a portion of the Vps2l-S21N mutant protein migrated as the prenylated 22 kda form. This raises the interesting possibility that the effector domain and GTP binding domain I of Vps21p is required for efficient geranylgeranylation. In fact, when the subcellular fractionation patterns of the GTP binding and effector domain mutant proteins were analyzed, the larger 23 kda nonprenylated form of these two proteins were found in a soluble cell fraction (S100) (data not shown). When the GTP binding capacity of the mutant Vps2l proteins were examined, both the T39 effector domain mutant and the S2 IN GTP binding motif I mutant were found to be defective for GTP binding (Figure 9C, lanes 1 and 2). The wild type Vps21p and the C208,2 OS prenylation site mutant protein were capable of binding similar amounts of GTP. Because the effector domain and GTP binding motif I mutants also interfere with prenylation, we cannot conclude whether the protein sorting defect results from the lack of GTP binding or from a decreased level of prenylation. In the case of the C208,2 OS prenylation site mutant that bound GTP normally, the defect in CPY sorting presumably reflects a defect in membrane association of this mutant protein as suggested by the subcellular fractionation experiments (Figure 8). 90 ": 80 N ce 70 E 60 0 < ' * 20 -' rt-* lo Time (min) Fig. 10. a-factor internalization in wild type and vps2l mutant strains. 35S-label az-factor was bound to wild type (filled square, GPY74-15C; VPS21 Bar-) cells, cells carrying the disrupted allele of vps2l (filled circle, BHY162; vps2j Bar-) or end3 mutant cells (filled triangle, RH266-ID Bar-) at 0 C. a-factor internalization was initiated and at the times indicated, equal aliquots were moved and washed with a ph 6.0 buffer (generating the value for total ai-factor, bound and internalized) or washed with a ph 1.1 buffer to remove any surface bound ax-factor (generating the value for internalized ca-factor). The percent a-factor internalized is presented as a function of time (min). Mating factor uptake is largely unaffected in vps21 mutant cells rab5 proteins have been shown to be involved in the endocytic pathway of mammalian cells (Chavrier et al., 1990; Gorvel et al., 1991; Bucci et al., 1992), yet in yeast we found that mutations in the rab5 homolog, VPS21, resulted in a severe vacuolar protein sorting defect. In order to determine whether vps2l mutants also exhibit defects in endocytosis, the uptake of the mating pheromone ca-factor was examined. a-factor normally enters the yeast cell via receptor-mediated endocytosis (Sprague and Thorner, 1992). By monitoring the internalization of radiolabeled a-factor, the competence of the early stages of the endocytic pathway can be monitored. s seen in Figure 10, vps2j mutant cells internalized a-factor at nearly wild type rates and at overall levels only slightly less than that of wild type cells. In contrast, a defined endocytosis mutant (end3; Raths et al., 1993) showed a severe defect in both the rate and overall levels of a-factor uptake. This result indicates that Vps2lp does not appear to serve an essential function in the initial stages of the endocytic pathway. However, this does not rule out the possibility that Vps2 lp is required at later stages of the endocytic pathway where the vacuolar protein sorting and endocytic pathways converge (Singer and Riezman, 1990; Vida et al., 1993). Discussion Vps21p exhibits all the characteristics of a ras-like GTP binding protein. The protein is the same size as ras, it contains highly conserved GTP binding motifs and an essential effector domain, it binds GTP, it is C-terminally prenylated, and it contains an intrinsic GTPase activity (B.F.Horazdovsky and S.D.Emr, unpublished data). Sequence comparisons show that, despite the general pe vps~vp21 / ^~~~~~~~~ed3 1304

9 GTP binding protein required for protein sorting homology shared with the superfamily of ras-like GTP binding proteins, Vps2lp is most closely related to the Sec4p/Yptl/rab members of this protein family (Balch, 1990; Pfeffer, 1992), especially rab5 (Chavrier et al., 1990; nuntalabhochai et al., 1991; rmstrong et al., 1993a). In mammalian cells, rab5 protein has been shown to be involved in the early endocytic pathway (Chavrier et al., 1990; Gorvel et al., 1991; Bucci et al., 1992). In yeast, the Vps21-rab5 homolog is required for vacuolar protein sorting. The loss of Vps21p function severely compromises the ability of yeast cells to localize both soluble and membrane-associated vacuolar proteins (Figure 5). Greater than 80% of the soluble hydrolases CPY and Pr are missorted in vps2j cells. The remaining -20% consists of a small portion of mature CPY and Pr as well as a larger amount of aberrantly processed forms of CPY and Pr. The appearance of these aberrant forms indicates that they may not be properly delivered to the vacuole but instead may reside in an intermediate compartment, such as the endosome or a transport vesicle. The maturation of a vacuolar membrane protein, LP, is also defective in vps2j mutant cells; > 70% of this protein accumulates as the Golgi-modified precursor form. Even after extended chase periods (60 min), the amount of precursor LP remains unchanged, indicating that most of the LP is missorted and not simply matured slowly in the vacuole. These results demonstrate that Vps2lp function is required for the efficient delivery/sorting of both membraneassociated and soluble vacuolar hydrolases. Because vps2j mutant cells still contain vacuoles, we assume that the small amount of mature CPY, Pr and LP detected in these cells is present in the vacuole and that this low level of sorting is sufficient to maintain vacuole integrity. Prenylation and membrane association of Vps21p The majority of Vps2 lp fractionates as a membraneassociated protein; >75% of Vps2 Ip pellets with a crude membrane fraction and can be extracted with detergent but not NaCl (Figures 6 and 7). In addition, the soluble nature of the C208,210S mutant Vps21p (lacking the prenylation site) and of wild type Vps2lp in bet2 mutant cells (lacking a subunit of the geranylgeranyl transferase) (Rossi et al., 1991; rmstrong et al., 1993b; Jiang et al., 1993) strongly indicates that C-terminal geranylgeranylation of Vps2 Ip is responsible for the membrane association of this protein (Figures 8 and 9). Vps2lp appears to be associated with at least two distinct membranes fractions (Figure 7). One pool of Vps2 Ip cofractionates with a P13 membrane fraction enriched in a number of cellular membranes, including vacuolar membranes; a smaller pool of Vps21p is associated with a high speed membrane pellet (P100) that includes Golgi membranes and small membrane vesicles (Walworth et al., 1989). nother GTP binding protein, Sec4p, has also been shown to associate with two distinct membrane fractions: a vesicular transport intermediate and the target membrane with which the vesicles fuse (Bruno et al., 1988). t this point, it is difficult to determine the exact composition of the two membrane pools with which Vps2lp associates. However, it seems reasonable to speculate that at least a portion of Vps2 ip may be associated with transport vesicles (P100) and a potential target membrane(s) (P13) that may include the vacuole and/or a prevacuolar endosomal membrane. Unfortunately, the yeast endosomal compartment is poorly characterized and no marker proteins are available to determine whether Vps2lp is associated with this membrane constituent. The apparent geranylgeranylation of Vps21p results in the conversion of Vps2ip from a protein that migrates with a molecular mass of 23 kda to one with a mass of 22 kda (Figure 5). The smaller form of Vps21p is associated with subcellular membranes (P13 and P100), whereas the larger form of the protein is found almost exclusively in the soluble/cytosolic fraction (S 100). In the C208,210S Vps2lp prenylation defective mutant or when wild type Vps2 Ip is examined in the bet2 mutant strain, only the larger soluble form is detected. These results indicate that the geranylgeranyl modification of Vps2lp results in the mobility shift (from 23 to 22 kda) and that this modification is required for the membrane association of Vps2ip (Figure 8). Further analysis of the post-translational modifications on Vps2lp will be required to determine whether the protein is subject to other modification/processing events in addition to geranylgeranylation. Intact GTP binding domain, effector domain and prenylation site are required for Vps2lp function To characterize further Vps2lp function, we analyzed the significance of one of the GTP binding motifs, the proposed effector domain and the prenylation site in Vps2lp using site-directed mutagenesis (Figure 9). Changes within these segments completely abolished Vps2 Ip function, leading to a severe vacuolar protein sorting defect and a growth defect at 38 C. This could not be attributed to instability of the Vps21 protein because each mutant protein was present at a level similar to that of wild type Vps2lp. In the case of the GTP binding domain mutant (S2 1N), the mutant Vps21 protein migrated on gels as both the small prenylated 22 kda form and the unmodified 23 kda form. Not unexpectedly, analysis of the GTP binding capacity of this mutant protein demonstrated that it was defective for GTP binding. Because much of this protein retains sufficient structural integrity to be recognized by the geranylgeranyl transferase, we suspect that the single S21N amino acid change specifically interferes with GTP binding. Interestingly, a mutation in the effector domain (T39) results in a mutant protein that does not appear to be a substrate for the geranylgeranyl transferase; only the larger 23 kda unmodified form of Vps21p was found in cells expressing this mutant protein. The T39 change could result in a global conformational change in the protein or the effector domain may serve some role in the association of Vps2lp with the geranylgeranyl transferase. recent report has indicated a role for the effector domain in the interaction of rab proteins with the geranylgeranyl transferase. rablb effector domain mutant protein is not prenylated by the geranylgeranyl transferase (Wilson and Maltese, 1993). Mutations corresponding to the GTP binding site mutation in VPS21 (S2 IN) have been shown to lead to dominantnegative phenotypes in ras and rab 1 (Freig and Cooper, 1988; Tisdale et al., 1992). In the case of the Vps21p mutation, no such dominant-negative phenotypes were seen even when the mutant Vps21 proteins were expressed at >20 times wild type levels. One possible explanation for this result is that because these mutant proteins are not geranylgeranylated as efficiently as wild type Vps2 Ip, they cannot bind to their target membrane and thereby interfere 1305

10 B.F.Horazdovsky, G.R.Busch and S.D.Emr Fig. 11. The possible sites of Vps2lp action in the yeast vacuolar protein sorting pathway. Due to the severity of the vacuolar protein sorting defect in vps2j cells, Vps2lp seems to function at one of two sites, the delivery of proteins from the Golgi to the late endosome or delivery from the late endosome to the vacuole. Due to the subcellular localization of Vps2lp and the accumulation of small nm transport vesicles, Vps2lp may function to promote the fusion of transport vesicles with their target membranes. with wild type Vps2lp function. Membrane association may be required to uncover the dominant-negative phenotype. Models for Vps21p function in vacuolar protein sorting The essential role of small GTP binding proteins in vesiclemediated protein transport has been well documented (Pryer et al., 1992). Based on these studies, the studies of lysosomal protein sorting in mammalian cells, and our observations regarding Vps21p function, we can propose a model for the role of Vps21p in vacuolar protein sorting (Figure 11). In a late Golgi compartment (perhaps equivalent to the trans- Golgi network), proteins destined for the vacuole are actively sorted away from the bulk of secretory proteins (Graham and Emr, 1991). This is mediated by the recognition of the sorting signals of soluble vacuolar proteins by transmembrane sorting receptors. CPY sorting receptor appears to be encoded by the VPSJO gene (E.G. Marcusson and S.D.Emr, unpublished data). The receptor-ligand complexes are packaged into vesicles and delivered to a late endosome where the ligand dissociates from the receptor and the receptor then recycles back to the Golgi to be utilized again for another round of transport (see Wilsbach and Payne, 1993). The vacuolar hydrolases would continue on to the vacuole probably via a membrane vesicle intermediate. The vacuolar/lysosomal protein sorting pathway and the endocytic pathway appear to converge at the late endosome in both mammalian cells and yeast (see ornfeld and Mellman, 1989 as well as Riezman, 1993 for reviews) Endocytosed proteins, like the pheromone a-factor and the pheromone receptors, are internalized and transported to the vacuole through early and late endosomal intermediates (Singer and Riezman, 1990; Vida et al., 1993). Vacuolar hydrolases and endocytosed proteins appear to share the last segment of the this delivery pathway; endocytosed a-factor and a portion of the p2 precursor form of CPY have been shown to cofractionate on sucrose density gradients (Vida et al., 1993). Our data are consistent with a model in which Vps21p functions either between the Golgi and late endosome or between the late endosome and the vacuole to facilitate vacuolar protein delivery. In addition, the accumulation of vesicles in vps2i mutant cells suggests that Vps2lp is specifically required for the targeting and/or fusion of these potential transport vesicles with their target membrane, the endosome or vacuole (Figure 11). nother small GTP binding protein, Ypt7p, has been demonstrated to function in the yeast endocytic pathway (Wichmann et al., 1992). Though the specific site of Ypt7p function is not completely clear, recent evidence indicates that it may play a role in the movement of proteins from a late endosomal compartment to the vacuole (Schimmoller and Riezman, 1993). The potential relationship between the pathways involving Ypt7p and Vps2lp is presently being examined. Using the dominant-negative mutants, antibody inhibition and immunofluorescent techniques, rab5 has been shown to play a role in the early stages of the mammalian endocytic pathway (Chavrier et al., 1990; Gorvel et al., 1991; Bucci et al., 1992). Recently, Zerial and his colleagues have isolated a set of three yeast genes that are predicted to encode proteins that share homology with mammalian rab5 proteins (M.Zerial and B.Singer-ruger, personal communication). One of these genes, YPT51, has been shown to be identical to VPS21. s shown here, disruption of the VPS21 gene results in severe vacuolar protein sorting defect and no major defect in a-factor internalization. However, the rate of a- factor degradation is dramatically delayed in this mutant (M.Zerial, personal communication). Since yeast endocytosis mutants that show defects in the early stages of the endocytic pathway do not affect vacuolar protein delivery (end3 and end4; Raths, 1993), the block in a-factor degradation seen in vps21 mutants is most consistent with Vps21p functioning in the movement of CPY and endocytosed proteins from a late endosomal compartment to the vacuole. However, the arrival of the endocytosed a-factor in the vacuole is biochemically scored by the degradation of this peptide by vacuolar proteases. If vacuolar proteases are not efficiently delivered to the vacuole (as is the case in the vps2i mutant strain), then the delay in degradation of oa-factor may result from the reduced levels of protease activity in this compartment. One of the best ways to address whether the above model is correct would be to isolate and characterize a temperature conditional vps21 mutant. In such a mutant, the vacuole could first be pre-loaded with active vacuolar proteases at the permissive temperature then the cells could be shifted to the nonpermissive temperature and the immediate effect of the loss of Vps2lp activity on the endocytic and biosynthetic sorting pathways could be monitored. We are currently attempting to construct such a vps2its mutant so that the precise stage at which Vps21p function is required in the vacuolar protein sorting/endocytic pathway can be identified. In addition, we hope to identify other accessory proteins that

11 GTP binding protein required for protein sorting Table I. Strains used in this study Strain Genotype Source S.cerevisiae SEY6210 MTo leu2-3,112 ura3-52 his3-200 trpl-901 lys2-801 suc2-9 Robinson et al. (1988) SEY6211 L4Ta leu2-3,112 ura3-52 his3-,200 trpl-90 ade2-101 suc2-l9 Robinson et al. (1988) BHY1O SEY6210 leu2-3,112::pbhyl 1(CPY-Inv LEU2) This study BHY1 1 SEY6211 1eu2-3, 12::pBHYl 1(CPY-Inv LEU2) This study BHY1O.5 M Ta/M Ta leu2-3,112::pbhyl 1(CPY-Inv LEU211eu2-3,112::pBHYl 1(CPY-Inv LEU2) his3-2001his3-200 ura3-521ura3-52 trpl-901/trpl-901 suc2-9isuc2-9 DE21ade2-101 lys2-8011lys2) This study SEY21-1 SEY6210; vps21-1 Robinson et al. (1988) SEY21-2 SEY62 10; vps2l-2 Robinson et al. (1988) NY1 19 MTa bet2-1 ura3-52 his4-619 Rossi et al. (1991) SFY26-6 MTax his4-619 Rossi et al. (1991) GPY74-15C MTa sstl-3 leu2-3,122 ura3-52 trpl-289 His- Greg Payne BHY160 SEY21-2; 1eu2-3,1 12::pBHYl l(cpy-inv, LEU2) This study BHY161 BHYII; VPS211VPS21::TRP1 This study BHY162 GPY74-15C; vps21j1::his3 This study GBY1O SEY6210; vps211::his3 This study GBY11 SEY62 11;, vps21j1::his3 This study GBY14 BHY1O.5; VPS211vps21J1::HIS3 This study RH266-1D MTa end3 ura3 teu2 his4 bar,-1 Raths et al. (1993) Ecoti JM101 D(lac-pro) supe thi-1 F' trad36 lactq ZM15 prob Yanish-Perron et al. (1985) MC1066 F- lacxyz hsr- hsm+ spsl galw gal trpc9830 leub600 pyrf::tns Martineza-rias and Casadaban (1983) JF1754 hsdr metb leub hisb lac gal McNeil and Friesen (1981) XL1-Blue supe44 thi-j lac endi gyr96 hsdrj7 retl F' prob laclq ZM15 TnlO Bullock et al. (1987) BMH71-18 muts Zhu (1992) regulate Vps2ip function (e.g. GTPase activating protein, nucleotide exchange factor and factors that direct Vps2lp to a specific membrane target). Mutations in genes encoding certain of these associated proteins may already be represented in the > 40 known vps complementation groups. These genetic and biochemical approaches should provide insights into the function of Vps2 Ip as well as possibly other small GTP binding proteins in vesicle-mediated protein transport events. Materials and methods Strains and media The S.cerevisiae and Escherichia coli strains used in this study are listed in Table I. Bacterial strains were grown on standard media (Miller, 1972). Yeast strains were grown in yeast extract, peptone, dextrose (YPD), yeast extract, peptone, fructose (YPF) medium or in synthetic medium (SM) supplemented as necessary (Sherman et al., 1979). In radiolabeling experiments, strains were grown in SM medium including 2% yeast extract prior to labeling. Yeast and bacterial methods Standard yeast genetic techniques (crosses, sporulation of diploids and tetrad dissection) were performed as previously described (Sherman et al., 1979). Yeast transformations were performed using the alkali cation treatment of Ito et al. (1983) and 5 itg of single stranded carrier DN per transformation (Schiestl and Gietz, 1989). E.coli transformations (JM101, MC1066, JF1754, XLI-Blue, BMH71-18) were performed using the method of Hanahan (1983). Yeast strains BHY10, BHYII and BHY160 were generated by integrating plasmid pbhyl 1 (CPY - invertase::leu2) at the leu2-3,122 locus of SEY6210, SEY6211 and SEY21-2 respectively. Integrative mapping studies of the cloned VPS21 gene were carried out by linearizing pbhy21-26 (VPS21, TRPI) at Sall and using the linearized plasmid to transform BHYl 1. Trp+ transformants (BHY161) were crossed with BHY160, diploids were selected, sporulated and the 22 of the resulting asci were dissected. Trp+/Trp- and Vps+/Vps- segregated 2:2. ll Trp+ haploid segregants also expressed the Vps' phenotype. To construct a chromosomal deletion mutant at the VPS21 locus, BHYIO.5, SEY6210 and SEY6211 were transformed with a CtaI-PvuII fragment of pgby21-4 (vps21::his3) (Figure IB) selecting for His' transformants, generating GBY14, GBY1O and GBYl 1 respectively. GBY14 was sporulated and 10 of the resulting asci were dissected. Genomic DN from representative haploid segregants as well as GBYlO and GBYl 1 were subjected to a PCR analysis to confirm the presence and site of the appropriate deletion/disruption (Herman and Emr, 1990). Cloning VPS21 SEY21-2 (vps2l-2 leu2-3,112) cells carrying a plasmid encoding a CPY - invertase fusion protein (pcyi50, UR3, CEN) (Johnson et al., 1987) were transformed with a plasmid-based yeast genomic DN library (LEU2, CEN) (kindly provied by Philip Hieter). Ura+/ Leu+ transformants were selected, replica-plated onto YPF medium and incubated overnight at 300C. solution containing 125 mm sucrose (Ultrapure, Boehringer Mannheim, Indianapolis, IN), 100 mm sodium acetate (ph 5.5), 10 jig/ml horseradish peroxidase, 8 units/ml glucose oxidase, 2 mm O-dianisidine, 0.5 mm N- ethyl maleimide was mixed with an equal volume of 3% bacto agar solution (at 50 C) and immediately poured over the replica-colonies. fter 5-15 min of incubation at room temperature, colonies secreting the CPY-invertase fusion protein turned brown (indicating a Vps- phenotype) and the colonies with internalized invertase activity remained white (Vps+). Vps+ colonies were streaked for individual colonies and retested. Plasmids conferring the Vps+ phenotype were isolated (pgby21-1), amplified in E.coli and used to transform SEY21-1 and SEY21-2 to confirm its complementing activity. Plasmid constructions and site-directed mutagenesis Plasmid pbhyl 1 was used to integrate a CPY - invertase gene fusion into a number of yeast strains. This plasmid was constructed by ligating a blunted EcoR.I-Pvull fragment containing the CPY-invertase fusion gene (CYI50) into a blunted HindII and the SmaI site of integrating plasmid prs305 (Sikorski and Heiter, 1989). Treating pbhyl 1 with EcoRI linearized the plasmid in the middle of the plasmid LEU2 locus and, once transformed, drove integration at the chromosomal leu2-3,112 locus. The VPS21 complementing plasmid pgby21-5 was constructed by ligating the ClaI -Bgtl fragment of pgby21-1 into the CENbased shuttle vector prs414 (Sikorski and Heiter, 1989). 21t-based plasmid expression vector of VPS

12 B.F.Horazdovsky, G.R.Busch and S.D.Emr was generated by ligating the same ClaI-BglII fragment into prs424 (pbhy21-28) (Sikorski and Heiter, 1989). The integrative mapping plasmid pbhy21-26 was constructed by ligating the ClaI-PvuHl fragment of pgby21-1 (Figure I) into the integrating vector prs304 (TRPI) in which the polylinker Sail site had been destroyed. Plasmid pgby21-4 was used to delete and disrupt the chromosomal VPS21 locus and was generated in a two-step process. The Sail polylinker site of pbluescripts (Stratagene) was eliminated and the ClaI-PstI fragment of pgby21-1 (containing VPS21) was ligated into the ClaI and PstI site of the modified pbluescripts vector. The coding region of VPS21 was removed by cutting this secondary construct with Sail and Bgll and replacing it with a DN fragment encoding HIS3 (Figure ib). The resultant plasmid pgby21-4 was treated with ClaI and PvuII to release the vps211::his3 fragment. This purified fragment was used to disrupt/delete the chromosomal VPS21 locus in the strains described above. Plasmids pgby21-2 and pgby21-3 were used in sequence analysis and were constructed as follows. The 3.2 kb ClaI-PstI fragment of pgby21-1 was ligated into the yeast shuttle vector prs414 to generate pgby21-2 and the 2.2 kb HindHII-SalI fragment was ligated into prs415 (Sikorski and Heiter, 1989) to generate pgby21-3. trpe- VPS21 gene fusion was constructed by ligating the 660 bp BstXI-BamHI fragment of pgby21-2 (with the BstXI site blunted) into the SmnaI-BamHI sites of trpe fusion vector pthl (Dieckmann and Tzagoloff, 1985) to generate pbhy The resultant gene fusion encoded amino acids of Vps2lp. Oligonucleotide-directed mutagenesis was carried out using denatured pgby21-2, a mutagenizing oligonucleotide, and a selection oligonucleotide as described in the Transformer Site-Directed Mutagenesis it (Clontech Laboratories, Inc., Palo lto, C). Plasmid pbhy21-1 carries the S21-N alteration, plasmid pbhy21-12 carries the T39- alteration, and the plasmid pbhy21-15 carries the C208S C210S double alteration. The appropriate amino acid changes in these plasmids were confirmed by sequence analysis. Sequence analysis of VPS21 Plasmids pgby21-2 and pgby21-3 were used to generate a set of exonuclease Im deletion mutants within the VPS21 complementing fragment. Deletion plasmids were size selected, denatured and purified using the Miniprep Plus procedure (Pharmacia, Inc., Piscataway, NJ) as described previously (Horazdovsky and Emr, 1993). The resultant denatured templates were hybridized to the T7 or T3 primers (Stratgene, Inc., La Jolla, C) and subjected to dideoxy chain termination sequence analysis (Sanger et al., 1977) using Sequenase sequencing protocol (US Biochemical Corp., Cleveland, OH). Second strand sequence analysis was performed using pgby21-2 and evenly spaced VPS21 derived olgionucleotides. The predicted amino acid sequence of VPS21 was compared with sequences available in the GenBank and EMBL databases using the BLST network service (ltschul et al., 1990; arlin and ltschul, 1990). Preparation of antiserum directed against Vps21p trpe- VPS21 gene fusion carried on plasmid pbhy21-20 was used to transform bacterial strain JMIOI. Protein production was induced and the Vps2l fusion protein was purified by the method of leid (leid et al., 1981) as modified by Herman and Emr (Herman and Emr, 1990). Purified fusion protein was used to immunize New Zealand White rabbits as previously described (Horazdovsky and Emr, 1993). The resulting antiserum was tested and titered by immunoprecipitation of labeled yeast cell extracts. Cell labeling, immunoprecipitations and subcellular fractionation Cells were grown in YNB medium containing 2% yeast extract and the appropriate amino acid supplements to an optical density at 600 nm (6W) of W units of cells were collected by centrifugation and suspended in 1 ml of YNB medium containing 1 mg/ml bovine serum albumin and 100 yci of Tran35S-label (ICN Radiochemicals, Costa Mesa, C). Cells were incubated at 30 C for the indicated times. When needed, a chase period was initiated by adding methionine and cysteine to a final concentration of 5 and 1 mm respectively. Yeast extract was also added during the chase period at a final concentration of 2%. Immunoprecipitations of the labeled yeast cell extracts were carried out as previously described (lionsky et al., 1988). CPY, Pr and LP fractionations were carried out as previously described (Horazdovsky and Emr, 1993). Subcellular fractionations of spheroplasts generated from wild type cells (SEY6210), SEY6210 carrying the VPS21 21s plasmid construct pbhy21-21 or GBY1O (vps21j1) carrying mutant VPS21 plasmids were labeled with Tran35S-label for 30 min and chased for 30 min at 30'C. The labeled spheroplasts were lysed and subjected to sequential centrifugation at 500 g (10 min), g (10 min) and g (60 min) as described by Horazdovsky and Emr (1993). lternatively, cleared cell lysates (500 g supernatants) were immediately subjected to centrifugation at g for 60 min. To determine the nature of the association of Vps21p with the pelletable material, cleared lysates (500 g supernatants) were adjusted to 1 M NaCl, 1% Triton X-100 or left untreated and incubated on ice for 10 min prior to centrifugation at g for 60 min. Subcellular fractionation of the bet2 mutant and wild type parental strains were also carried out as above except spheroplasts generated from these strains were first preincubated for 15 min at 37 C prior to a 10 min labeling period and a 30 min chase at 37 C. The presence of Vps2 lp, glucose-6-phosphate dehydrogenase (G6PDH), LP or ex2p in these subcellular fractions were determined by immunoprecipitation as previously described (Horazdovsky and Emr, 1993). Western and GTP binding analysis Western and GTP binding analysis of wild type and mutant Vps2lp were carried out by first immunoprecipitating the various forms of Vps2 Ip from 5 6W equivalents of unlabeled yeast cell extracts generated by glass bead lysis. Equal amounts of immunoprecipitates were resolved using SDS-polyacrylamide electrophoresis. The acrylamide gels were processed and the proteins transferred to nitrocellulose filters as described by Wagner et al. (1992). One blot was processed for Western analysis (Dunn, 1986) using anti-vps2lp serum and goat anti-rabbit horseradish peroxidase conjugated secondary antiserum as described in ECL Western blotting protocols (mersham International). duplicate blot was analyzed for Vps2lp [a-32p]gtp (mersham International) binding as described by Wagner et al. (1992). a-factor internalization The binding and internalization of 35S-labeled a-factor in wild type and vps211 cells (GBY1O) was determined using the procedure of Dulic et al. (1991) as modified by Tan et al. (1993). Briefly, 35S a-factor was bound to 200 6W of cells in 2 ml of ice-cold 50 mm potassium phosphate (ph 6.0) and 1% bovine serum albumin c.p.m. of labeled a-factor was added per 100 1il of cell suspension and binding was allowed to proceed for 30 min with shaking at 0'C. Cells were pelleted (500 g for 10 min at 0 C ) and suspended in potassium phosphate buffer + bovine serum albumin as above. One-hundred microliter aliquots of cell suspension were removed and warmed to 25 C for 5 min. Glucose was then added to 2% to initiate internalization. fter various incubation times the 100,ul samples were placed in 10 ml of ice-cold internalization buffer [50 mm sodium citrate (ph 1.1)] and incubated on ice for 15 min. Duplicate aliquots were placed in 10 ml ice-cold 50 mm potassium phosphate buffer (ph 6.0). Cells were collected by filtration and processed as described by Dulic et al. (1991). Electron microscopy analysis SEY6210 (VPS21) and GBY1O (vps2jj) were grown in YPD medium at 30 C to an 6W of 0.5. Cells were fixed directly in the growth medium by adding glutaraldehyde to 2%, CaCl2 to 5 mm and cacodylate (ph 6.8) to 0.1 M equivalents of fixed cells were harvested by centrifugation, resuspended in 1 ml of 3% glutaraldehyde, 5 mm CaCl2 and 0.1 M cacodylate (ph 6.8), and incubated for 1 h at 25 C. Cells were prepared for embedding as described (Banta et al., 1988). Cells were then embedded in low viscosity Spurr plastic resin for 48 h at 60'C. Sections were stained with Reynold's lead citrate for 2 min and 2% uranyl acetate for 10 min at 25 C. Cell sections were viewed at 80 kv using a JOEL 12 EX transmission electron microscope. cknowledgements We thank the Emr laboratory for critically reading the manuscript and for many helpful discussions during the course of this work. We would also like to thank Marino Zerial and Birgit Singer-ruger for communicating results prior to publication, Susan Ferro-Novick for supplying the bet2 strain, Phil Tan and Greg Payne for providing us with labeled ai-factor, Bill Wickner and Elizabeth Jones for supplying ex2p and PrB antisera, as well as Micheal McCaffery for his help with the electron microscopy. This work was supported by a grant from the National Institutes of Health (GM-32703) and the National Cancer Institute (C58689). S.D.E. is supported as an investigator of the Howard Hughes Medical Institute. 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