INTRODUCTION

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The intracellular compartments of eukaryotic cells are largely defined by the composition of the resident proteins contained therein. Thus, efficient biosynthetic trafficking of these resident proteins must be maintained to ensure the integrity and functionality of individual organelles. The acidified vacuole of the budding yeast Saccharomyces cerevisiae is one such organelle. Analogous to the lysosome of mammalian cells, the vacuole functions in a variety of cellular processes, including macromolecular degradation, metabolite storage, and cytosolic ion and pH homeostasis (41, 45). Thus, vacuolar function requires an influx of resident proteins such as hydrolytic proteases, lipases, and transporters. These proteins traffic via vesicle-mediated transport reactions that require appropriate cargo selection and vesicle budding from donor membranes, followed by the docking and fusion of the transport intermediates with the correct target organelle. Several genetic screens have been designed to identify mutants defective in vacuolar protein sorting (vps) (4, 68), vacuolar protease activity (pep) (40), and vacuolar morphogenesis (vam) (84). Through these screens, over 40 complementation groups have been identified that display defects in the delivery of proteins from the trans-Golgi network to the vacuole. These complementation groups have been further classified (classes A through F) with respect to their specific morphological and biochemical phenotypes (5, 63).

Biochemical investigation of protein transport in numerous experimental systems has identified several proteins proposed to be involved in the docking and fusion of transport vesicles with their target membranes (30, 66, 67, 78). These proteins have been found to be highly homologous among the various systems, suggesting that the docking and fusion of transport vesicles utilizes a conserved mechanism (11, 23). Namely, members of the syntaxin, synaptobrevin-VAMP, Sec1p, and Rab families have been shown to function in vesicular transport at multiple stages of the secretory pathway. In neuronal cells, the transmembrane proteins synaptobrevin (v-SNARE) and syntaxin (t-SNARE) associate with donor and target membranes, respectively (8, 10, 79). According to the SNARE hypothesis, complementary pairing between synaptobrevin and syntaxin provides, in part, the specificity required for the targeting of cargo to the appropriate destination (76). Stable interaction between synaptobrevin and syntaxin requires an additional t-SNARE molecule, SNAP-25 (synapse-associated protein of 25 kDa), that forms a t-SNARE complex with syntaxin (31, 32, 58, 75). This ternary interaction ensures the docking stability and specificity necessary for subsequent fusion to occur. Sec1p and Rab family members are suggested to regulate the formation and/or activity of these SNARE interactions (56, 66, 67). After the coupling of v- and t-SNAREs, N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) catalyze the disassembly of SNARE complexes, allowing fusion of the transport vesicle with the target membrane (16, 75, 76). Alternatively, it has also been proposed that the NSF-SNAP complex functions in priming SNAREs via ATP hydrolysis prior to docking (50, 51, 55, 80).

Several of the VPS gene products have been found to be members of the conserved syntaxin, Sec1p, and Rab families. The syntaxin homolog Pep12p (9), Sec1p family member Vps45p (17, 60), and Rab GTPase Vps21p (35) are believed to direct the transport of vacuolar proteins from the trans-Golgi to an endosomal compartment (9, 15). Genetic and biochemical studies have also identified a set of VPS gene products that mediate trafficking at a late stage of vacuolar protein transport. These gene products include Vam3p (20, 77, 83), Vps33p (6, 65), and Ypt7p (86), which share homology with syntaxin, Sec1p, and Rab family members, respectively. Additionally, the yeast NSF homolog Sec18p (21) has been shown to function both in Golgi-to-endosome transport with Pep12p (15) and in vacuole-to-vacuole fusion with Ypt7p and Vam3p (50, 55, 80). The prevalence of these SNARE complex members suggests that the mechanisms of docking and fusion are conserved in the vacuolar protein sorting pathway.

The VAM7 gene was originally identified in a screen for mutants defective in vacuolar assembly and morphogenesis (84). Cells with the VAM7 gene deleted lack normal vacuoles and, instead, accumulate numerous vesicular structures that contain vacuolar proteins (82). Furthermore, Vam7p has been determined to contain an amino-terminal NADPH oxidase p40^phox (PX) domain (61) and a carboxy-terminal heptad repeat homologous to the coiled-coil domain of SNAP-25 family members (85). Here, we report the function of the VAM7 gene product in vacuolar protein sorting by characterizing a temperature-conditional allele of VAM7 (vam7^tsf). Analysis of the vam7^tsf mutant indicates that Vam7p functions at a late stage in vacuolar delivery of multiple proteins that transit via distinct biosynthetic pathways. Inactivation of the vam7^tsf gene product results in the accumulation of numerous aberrant membrane compartments which likely correspond to unfused transport intermediates. Localization experiments suggest that a fraction of Vam7p associates with vacuolar membranes. Genetic and physical analysis determined that Vam7p functionally interacts with the vacuolar t-SNARE Vam3p. Our results suggest that Vam7p functions at the vacuole as part of a t-SNARE complex in a manner analogous to SNAP-25 in synaptic vesicle trafficking.

	MATERIALS AND METHODS

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Strains and media. The S. cerevisiae strains used in these studies are listed in Table 1. Yeast strains were grown in standard yeast extract-peptone-dextrose (YPD), yeast extract-peptone-fructose (YPF), or synthetic dextrose (SD) media supplemented with appropriate amino acids (72). Yeast transformations were done by the lithium acetate method (39) with single-stranded carrier DNA. Standard bacterial medium (52) was supplemented with 100 µg of ampicillin per ml for plasmid retention. Escherichia coli transformations with the XL1Blue strain (14) were done as previously described (28).

DNA methods. Recombinant DNA manipulations were performed by standard methods (49). Restriction and modification enzymes were purchased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and New England Biolabs (Beverly, Mass.). DNA sequencing was done by the UCSD CFAR Core facility with the ABI Prism BigDye and Amplitaq DNA polymerase FS. Gels were analyzed with a Perkin-Elmer-Applied Biosystems 373XL DNA sequencer. The plasmid pYVQ706 containing the VAM7 open reading frame was a generous gift from Yoh Wada (University of Tokyo, Tokyo, Japan). Its construction is described elsewhere (82). Plasmids pTKS30 and pTKS23 were generated by subcloning the 1.8-kb NsiI-HindIII fragment (containing the entire VAM7 ORF) of pYVQ706 into the PstI-HindIII polylinker sites of pRS414 and pRS424 (73), respectively. A PCR-based HIS3 disruption fragment was constructed with primers containing overhangs of the first and last 50 bases of the VAM7 coding sequence (7). The amplified DNA fragment was isolated and transformed into SEY6210 or BHY10. The resulting His⁺ colonies were isolated and confirmed for insertion of the HIS3 gene into the VAM7 locus by PCR of genomic DNA. Furthermore, this procedure was used to generate other vam7 strains described in Table 1. Plasmid pTKS43ts-167 containing a temperature-conditional allele of vam7 (vam7-167) was constructed by PCR-based mutagenesis and gapped-plasmid repair (53). Primers complementary to the 5' or 3' ends of the VAM7 coding sequence were used to mutagenize VAM7 under limiting dATP conditions. The resulting PCR product was purified and cotransformed with PstI-AatII gapped pTKS30. Candidates were isolated for temperature-conditional secretion of a CPY-invertase fusion protein (57). The GFP-Vam7p fusion protein was constructed by PCR with oligonucleotides introducing an in-frame BamHI site at the start ATG. The PCR fragment was cloned into the BglII-SalI sites of the pRS416 version of pGOGFP (18) to generate plasmid pTKS35. PX domain point mutants were made by using the gene SOEing method (38, 87) and gapped-plasmid repair. Point mutations were recovered from yeast cells and confirmed by sequencing for the desired mutations. Plasmids pVAM3.414, pVAM3.416, pVAM3.426, pVAM3-6.416, and pPEP12.426 were previously described (20).

Preparation of antiserum against Vam7p. To construct the glutathione S-transferase (GST)-Vam7p fusion protein, the 943-base XbaI-HindIII fragment of VAM7 was inserted into PGEX-KG (Pharmacia LKB, Uppsala, Sweden). The resulting fusion protein containing the C-terminal 182 amino acids of Vam7p was expressed in E. coli XL1Blue and isolated by affinity binding to glutathione-coupled Sepharose. After being eluted from the Sepharose, the GST-Vam7p fusion protein was further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroelution. Purified protein was then used to immunize New Zealand White rabbits as previously described (36). Crude antiserum was affinity purified by binding to cyanogen bromide-coupled GST-Vam7p (29). Vam7p was detected in yeast cell lysates by immunoblotting and enhanced chemiluminescence (ECL) detection as previously described (2).

Metabolic labeling and immunoprecipitations. To examine the biosynthetic trafficking of vacuolar proteins, yeast cells were radiolabeled as previously described (2, 35, 36). Immunoprecipitations of ALP, CPS, and CPY were done accordingly (19, 36). Aminopeptidase I (API) immunoprecipitations were performed as previously described (20). API antibody was a generous gift from Dan Klionsky (University of California, Davis, Calif.) (43). Antibodies to ALP, CPY, CPS, Pep12p, and Vam3p have been previously described (9, 19, 20, 42, 44).

Subcellular fractionation. Subcellular fractionation experiments with Vam7p were done as previously described with modifications. Unlabeled spheroplasts made from wild-type cells (SEY6210) were lysed, and fractions were generated as described previously (24). Each fraction was detected by immunoblotting and ECL as described previously (2). For temperature-shift experiments, spheroplasts from EGY1181-10 were radiolabeled at 26°C with EXPRE³⁵S³⁵S label (NEN/DuPont) for 35 min and chased for 10 min with unlabeled methionine, cysteine, and yeast extract to final concentrations of 5 mM, 1 mM, and 0.2%, respectively. After 10 min, cells were either shifted to 38°C or remained at 26°C for an additional 30 min. After the temperature shift, cells were harvested, fractions were generated, and Vam7p was immunoprecipitated as described previously (24). Accudenz density gradients were prepared as described earlier (3). The 13,000 × g pellet fraction was generated from unlabeled spheroplasts as described above and resuspended in 1 ml of lysis buffer (0.2 M sorbitol; 50 mM potassium acetate; 20 mM HEPES, pH 6.8; 2 mM EDTA) containing 60% Accudenz A.G. (Accurate Chemical & Scientific Corporation, Westbury, N.Y.). A 2-ml portion of 55% Accudenz A.G. was then layered on top of the resuspended pellet, followed by an additional 2 ml of 35% Accudenz A.G. After a 14-h spin at 200,000 × g, the top 3 ml, the remaining 2 ml, and the sediment were individually harvested, precipitated with 10% trichloroacetic acid (TCA), washed with acetone, and detected by immunoblotting. The top 3 ml was designated the float fraction, and the remaining 2 ml was designated the nonfloat fraction.

Microscopy. Nomarski optics analysis and fluorescence microscopy of the green fluorescent protein (GFP)-Vam7p fusion protein were done as described previously (18). For electron microscopy (EM) analysis, vam7 mutants were prepared as described earlier (20, 64).

Native immunoprecipitations. Appropriate yeast strains were grown in YPD or SD supplemented with 2% Casamino Acids and appropriate amino acids to mid-log phase. Six units of optical density at 600 nm (OD₆₀₀; 1 OD₆₀₀ unit  10⁸ cells) were harvested, spheroplasted, and allowed to recover for 10 min at 26°C. Accordingly, the cells were then shifted to 38°C or retained at 26°C for 1 h and then lysed in 0.5% Triton X-100, 0.2 M sorbitol, 50 mM potassium acetate, 20 mM HEPES (pH 6.8), and 2 mM EDTA and homogenized as described previously (24) at 5 OD₆₀₀ units/ml. The homogenate was extracted on ice for 10 min and then centrifuged at 13,000 × g for 10 min. One milliliter of extract was transferred to a new Eppendorf tube and incubated with 5 µl of affinity-purified anti-Vam7p polyclonal antibody for 1 h at 4°C. Protein A-Sepharose beads (Pharmacia) were added, and the mixture was allowed to incubate for an additional 1 h. After incubation, bound protein was washed six times with 1 ml of lysis buffer containing 0.1% Triton X-100. Supernatant was completely removed from the beads with a Hamilton syringe and immunoprecipitated proteins were eluted with SDS-PAGE protein sample buffer. Two OD units were loaded, resolved by SDS-PAGE, and detected by immunoblotting and ECL.

	RESULTS

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A vam7^tsf mutant displays defects in the maturation of multiple vacuolar proteins and accumulates aberrant membranous compartments. Mutations in a specific subset of VPS and VAM genes cause an accumulation of vacuolar proteins in numerous small, prevacuolar compartments (5, 63, 84). Not surprisingly, many of the VAM genes overlap with the VPS genes, including VPS41/VAM2 and VAM6/VPS39 (54). A previous study determined that vam7 mutants also display a similar aberrant vacuolar morphology (82). In complementation studies, we found that the VAM7 gene complemented vps43 mutants, indicating that VAM7 and VPS43 correspond to the same locus. To address the primary role for VAM7, a temperature-conditional allele of VAM7 was generated by error-prone PCR mutagenesis as previously described (see Materials and Methods). One mutant, vam7-167, was characterized in detail. DNA sequencing analysis of the vam7-167 mutant identified two amino acid changes: leucine 134 to proline and leucine 287 to proline. Western blotting revealed that mutant Vam7p expression in the vam7-167 strain was found to be similar to that observed for wild-type Vam7p at both permissive and nonpermissive temperatures for 30 min (data not shown). Henceforth, this allele will be denoted as vam7^tsf (temperature-sensitive for function).

View larger version (58K): [in this window] [in a new window]   FIG. 1.   Vacuolar protein sorting in vam7tsf mutant cells. (A) TKSY43 (vam7) cells transformed with either complementing plasmid (pTKS30) or plasmid containing a temperature-sensitive-for-function (tsf) allele of vam7 (pTKS43ts-167) were incubated at either the permissive (26°C) or the nonpermissive (38°C) temperature for 10 min and then labeled with [35S]cysteine and [35S]methionine for 10 min. Cells were subsequently chased with unlabeled cysteine and methionine and harvested at the time points indicated. ALP, CPY, and CPS were immunoprecipitated, resolved by SDS-PAGE, and visualized by autoradiography. CPS samples were treated with endoglycosidase H prior to electrophoresis. ER-modified and Golgi-modified CPYs are denoted by p1CPY and p2CPY, respectively. Other precursor (p) and mature (m) forms of vacuolar proteins are indicated. (B) API delivery in vam7tsf cells. TKSY43 (vam7) cells transformed with either complementing plasmid (pTKS30) or plasmid containing the vam7tsf allele (pTKS43ts-167) were incubated at the nonpermissive temperature (38°C) for 10 min and then labeled with [35S]cysteine and [35S]methionine for 10 min. Cells were subsequently chased with unlabeled cysteine and methionine and harvested at the indicated time points. API was recovered from lysates by immunoprecipitation, resolved by SDS-PAGE, and visualized by autoradiography. Precursor API (prAPI) and mature API (mAPI) are indicated.

View larger version (138K): [in this window] [in a new window]   FIG. 2.   Ultrastructural analysis of vam7 and vam7tsf mutants. A cross-section of (A) wild-type (SEY6210) and (B) vam7 cells grown at 30°C viewed by EM. Mutant cells containing the vam7tsf allele (TKSY43 plus pTKS43ts-167) were incubated at the permissive temperature (26°C) or the nonpermissive temperature (38°C) for 3 h (C and D, respectively) prior to preparation for EM analysis. vam7tsf cells at the nonpermissive temperature accumulated novel compartments in addition to a prominent electron-dense vacuolar compartment. Enlargements of novel compartments (E) accumulated after 3 h at the nonpermissive temperature, including those that resemble multi-vesicular bodies (F and G, arrows) also seen in vam3tsf and vps41tsf mutants. n, nucleus; v, vacuole. Bars: A to D, 500 nm; E to G, 200 nm.

Vam7p associates with vacuolar membranes. To identify the VAM7 gene product, a polyclonal antiserum was raised against a fusion protein containing GST fused to the carboxy-terminal 182 amino acids of Vam7p. The resulting antiserum was affinity purified and specifically recognized a protein of ca. 35-kDa in wild-type cells that was not observed with preimmune sera or in vam7 cells (data not shown). This closely corresponds to the predicted molecular mass of 38.6 kDa for the product of the VAM7 open reading frame.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Subcellular localization of the VAM7 gene product. (A) SEY6210 (wild-type) spheroplasts (5 OD600 units) were lysed and fractionated by differential centrifugation (see Materials and Methods), generating P13, P100, and S100 fractions. Fractions were TCA-precipitated, and proteins from 2 OD600 units were resolved by SDS-PAGE. Resolved proteins were then transferred to nitrocellulose, immunoblotted with anti-Vam7p antibody, and visualized by ECL fluorography. EGY118-110 (sec18-1) spheroplasts were labeled with [35S]cysteine and [35S]methionine at the permissive temperature (26°C) for 30 min and chased for 15 min. Cells then remained at the permissive temperature or were shifted to the nonpermissive temperature (38°C) for an additional 30 min. After the temperature shift, cells were harvested and fractionated as described above. TCA-precipitated lysates were immunoprecipitated with anti-Vam7p antibody, revolved by SDS-PAGE, and visualized by autoradiography. (B) Accudenz gradient analysis of Vam7p from the P13 fraction of inactivated sec18-1 cells. The P13 fraction from EGY118-110 (sec18-1) cells incubated at the nonpermissive temperature (38°C) was resuspended in lysis buffer containing 60% Accudenz and loaded at the bottom of a 60%-55%-35% Accudenz step gradient. After centrifugation at 200,000 × g for 14 h, floating (F), nonfloating (NF), and pellet (P) fractions were harvested and analyzed by Western blotting.

View larger version (40K): [in this window] [in a new window]   FIG. 4.   Localization of the GFP-Vam7p fusion protein. (A) Complementation of the CPY and ALP sorting defects of vam7 mutants with the GFP-Vam7p fusion protein. TKSY43 (vam7) cells harboring a single-copy (CEN) plasmid (pTKS35) encoding the GFP-Vam7p fusion protein were labeled with [35S]cysteine and [35S]methionine for 10 min at 30°C and chased with unlabeled cysteine and methionine for the indicated times. CPY and ALP were immunoprecipitated and then visualized by SDS-PAGE and autoradiography. (B) Nomarski optics image (left panel) and fluorescence localization (right panel) of GFP-Vam7p in TKSY43 (vam7) cells containing the GFP-Vam7p fusion protein (pTKS35).

VAM7 interacts with the putative vacuolar t-SNARE VAM3. The VAM3 gene product has been shown to be required at a late step in the transport of multiple proteins to the vacuole. Similar to vam7^tsf mutants, a vam3^tsf strain displays defects in the transport of ALP, CPS, CPY, and API to the vacuole (20). Vam3p localizes to vacuolar membranes, and its homology to syntaxin suggests that it functions as a t-SNARE in protein transport to the vacuole (20, 77, 83). Similarities in phenotypes between vam3 and vam7 mutants together with their homologies to syntaxin and SNAP-25, respectively, suggest that they may function together at a common step in the VPS pathway.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Genetic interactions between VAM7 and VAM3. (A) Suppression of vam7tsf and vam3tsf mutant cells. TKSY43 (vam7) or TDY2 (vam3) containing tsf alleles of either vam7 (pTKS43ts-167) or vam3 (pVAM3.6-416), respectively, were transformed with a multicopy vector (2µm) containing no insert, VAM7 (pTKS23), or VAM3 (pVAM3.426) as indicated. Cells grown at 26°C were harvested, incubated at the permissive or nonpermissive temperature for 10 min, and labeled with [35S]cysteine and [35S]methionine for 10 min. After the labeling, cells were chased with unlabeled cysteine and methionine for 30 min and harvested by TCA precipitation. Lysates from these cells were immunoprecipitated with the indicated antibodies and analyzed as previously described. (B) Synthetic interactions between vam7tsf and vam3tsf. Double-mutant strains (vam3 vam7) harboring the vam3tsf allele (pTKS30 and pVAM3.6-416), the vam7tsf allele (pTKS43ts-167 and pVAM3-416), or both tsf alleles (pTK43ts-167 and pVAM3.6-416) were incubated at 26°C for 10 min and labeled with [35S]cysteine and [35S]methionine for an additional 10 min. After the labeling, the cells were chased with unlabeled cysteine and methionine for 45 min, harvested, and assayed for vacuolar protein sorting as described above. (C) Double-mutant cells (pep12-60 vam7) were transformed with CEN plasmids containing VAM7 (pTKS30) or the vam7tsf allele (pTKS43ts-167). Single- and double-mutant strains were assayed for CPY sorting as described above.

Vam3p coimmunoprecipitates with Vam7p. Our genetic findings suggest that Vam7p physically interacts with Vam3p in a complex on vacuolar membranes. In addition, the data suggest that this interaction may be enhanced by inactivation of the SEC18 gene product. In order to determine whether Vam7p may be in a complex that contains Vam3p, Vam7p was immunoprecipitated under native conditions with detergent extracts from wild-type and sec18-1 yeast cells. After immune complexes were pelleted with protein A-Sepharose beads, bound protein was washed with 0.1% Triton X-100 and then eluted from the beads with sample buffer. Immunoprecipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-Vam3p or anti-Vam7p antibodies (Fig. 6). When compared to a control fraction (lane 6), ca. 10 to 15% of total Vam3p coimmunoprecipitated with Vam7p in wild-type cells incubated at 38°C or sec18-1 cells incubated at 26°C (lanes 2 and 3). In striking contrast, in sec18-1 cells incubated at 38°C, the amount of Vam3p that coimmunoprecipitated with Vam7p increased ca. fivefold (lane 4). Importantly, Vam3p did not coimmunoprecipitate with Vam7p in extracts made from sec18-1 vam7 or sec18-1 vam3 strains incubated at 38°C (lanes 1 and 5). The interaction between Vam7p and Vam3p was also specific, as Pep12p did not coimmunoprecipitate with Vam7p in extracts made from sec18-1 cells incubated at 38°C (data not shown). Consistent with results from the genetic studies, these coimmunoprecipitation experiments demonstrate that Vam7p is in a complex that contains Vam3p.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Native immunoprecipitation of Vam3p with Vam7p. Spheroplasts (5 OD600 equivalents) from the following strains were generated and incubated at either 26 or 38°C as indicated for 1 h: TKSY48 (sec18-1 vam7), SEY6210 (wild type), EGY118-110 (sec18-1), and CBY42 (sec18-1 vam3). Spheroplasts were then lysed with 0.5% Triton X-100 and incubated with anti-Vam7p antibody for 1 h at 4°C, followed by the addition of protein A-Sepharose for an additional 1 h. Bound immunocomplexes were washed with 0.1% Triton X-100 and eluted from beads with SDS-PAGE sample buffer. Two-OD600-unit samples were loaded onto SDS-PAGE gels, resolved by electrophoresis, and transferred to nitrocellulose paper. Vam3p and Vam7p were immunoblotted with anti-Vam3p and -Vam7p antibody, respectively, and visualized by ECL fluorography. Of the total sec18-1 38°C extract, 5% was TCA precipitated and loaded as an indication of the amount of total Vam3p bound to Vam7p.

Mutations in the PX domain of Vam7p result in synthetic defects with the vam3^tsf mutant. Recently, an extensive database search revealed that Vam7p contains a domain that is present in a number of proteins with diverse functions (61). This domain, termed the PX domain, characterizes a family of proteins that includes the p40^phox and p47^phox subunits of the NADPH oxidase complex, CPK-like phosphatidylinositol 3-kinases, and proteins involved in vesicular trafficking, including the SNX-1 family members (47) and the yeast proteins Mvp1p (22), Vps5p (37), and Vps17p (46). No function has been determined for this domain, nor has any requirement for the PX domain in a specific protein been identified. The region of the PX domain in Vam7p with highest homology to Vps5p, Mvp1p, and Vps17p spans amino acids 26 to 89 (Fig. 7A). To determine whether the PX domain is critical for Vam7p function, two point mutations in codons of conserved amino acids were made: tyrosine 42 to alanine (vam7^Y42A) and leucine 48 to glutamine (vam7^L48Q).

View larger version (15K): [in this window] [in a new window]   View larger version (37K): [in this window] [in a new window]   FIG. 7.   Vacuolar trafficking defects of VAM7 PX domain mutants with the vam3tsf mutation. (A) The PX domain of Vam7p aligned with the PX domains of three yeast proteins involved in vesicular trafficking are shown. The regions of highest homology between the four PX domains are indicated by the numbers on the right and left of the sequence. Gap lengths are indicated in parentheses. Amino acid residues of at least two other proteins that are conserved with the PX domain of Vam7p are highlighted in black. Amino acid residues that are similar to Vam7p PX domain residues are indicated in gray. The amino acids that were changed (Y42A and L48Q) are denoted in boldface above the Vam7p sequence. (B) Synthetic interactions between vam7 PX and vam3tsf mutations. Standard pulse-chase analysis was conducted on vam3 vam7 double mutants harboring CEN plasmids containing wild-type VAM7 (pTKS30), mutant VAM7 encoding tyrosine 42-to-alanine (Y42A) or leucine 48-to-glutamine (L48Q) mutations, and wild-type VAM3 (pVAM3.416) or vam3tsf (pVAM3-6.416) alleles. Double mutants were labeled for 10 min and chased for 30 min at 26°C.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The experiments described here investigate the role of Vam7p in vacuolar protein sorting. Previous reports suggested that Vam7p functions in vacuolar morphogenesis (82) and identified an amino-terminal PX domain of unknown function (61) and a carboxy-terminal heptad repeat with homology to the coiled-coil of SNAP-25 (85). Analysis of the vam7^tsf mutant (double mutant; leucine 134 to proline and leucine 287 to proline) revealed that, like the vacuolar syntaxin homolog Vam3p (20), Vam7p functions in the delivery of multiple proteins that all converge at the vacuole via distinct biosynthetic pathways. Furthermore, examination of the vam7^tsf mutant incubated at the nonpermissive temperature by EM revealed the accumulation of numerous small aberrant membranous compartments, suggesting that the docking and/or fusion of transport intermediates to the vacuole was impaired. Localization experiments determined that a portion of Vam7p associates with vacuolar membranes. The large cytoplasmic pool and the absence of any consensus palmitoylation motif or shift in the electrophoretic mobility of the membrane-associated fraction suggest that Vam7p is not modified with palmitate as is SNAP-25 (33). However, transient palmitoylation cannot be ruled out. Genetic studies demonstrated that VAM7 functionally interacts with VAM3. Consistent with these observations, Vam7p was found to physically associate in a complex that contains Vam3p and this interaction was enhanced by inactivation of Sec18p. This suggests that Vam7p may be a component of a t-SNARE complex analogous to syntaxin-SNAP-25 complexes at the plasma membrane of neuronal cells. Lastly, mutations in the PX domain of Vam7p resulted in synthetic protein sorting defects when expressed in the vam3^tsf mutant. Together, the data presented here suggest that Vam7p functions in conjunction with Vam3p, possibly as part of a t-SNARE complex on vacuolar membranes to mediate the docking and fusion of multiple transport intermediates from distinct biosynthetic pathways.

Vam7p regulates a late step in vacuolar protein trafficking. Initial analysis of the VAM7 gene suggested it functions in the maintenance of vacuolar morphology but not in vacuolar protein sorting (84). Examination of vam7 mutants revealed that these mutants lack normal vacuoles and instead accumulate numerous abnormal membrane compartments. Analysis of CPY by Western blotting in vam7 mutants found CPY predominantly in its mature form (82). However, steady-state analyses of this kind can be misleading, as slow processing of vacuolar precursors can occur in nonvacuolar compartments. For example, nonvacuolar processing has been observed in Class E vps mutants which develop a proteolytically competent prevacuolar compartment (2, 59). Pulse-chase and EM analysis of the vam7^tsf mutant suggests that Vam7p functions primarily in protein trafficking. After temperature inactivation, while vacuolar integrity and inheritance remain normal, newly synthesized p2CPY immediately accumulates and is slowly processed to the mature form only after prolonged chase. Additionally, the bulk of p2CPY is not secreted, indicating that it is sorted away from the late secretory pathway at the trans-Golgi but may accumulate in transport intermediates, such as prevacuolar endosomes. This phenotype has also been observed in vps41^tsf (19) and vam3^tsf mutants (20). The common phenotypes associated with these vam and vps mutants suggest that they function together to regulate a late step in vacuolar transport.

Vam7p as a component of a vacuolar SNARE complex. Sequence similarities between some of the VPS gene products and proteins implicated in the docking of transport vesicles to target membranes in mammalian systems suggest that they share conserved functions. The identification of syntaxin homologs, Sec1p family members, and Rab GTPases that are required in vacuolar protein sorting implies that transport within the VPS pathway is mediated by mechanisms common to other vesicle-mediated transport steps. In neuronal cells, docking is mediated by the pairing of synaptobrevin (v-SNARE) on the vesicle membrane with the t-SNAREs syntaxin and SNAP-25 on the target membrane to form the stable complex necessary for fusion events (31, 32, 58, 76). Analysis of the Vam7p sequence identified a region (amino acids 253 to 313) predicted to form coiled-coil interactions that shares homology to SNAP-25 (85), suggesting that it may function in a similar manner.

The PX domain is required for Vam7p function. The PX domain is common to a diverse set of proteins, including two subunits of the NADPH oxidase complex, p40^phox and p47^phox, the CPK family of phosphatidylinositol 3-kinases, and phospholipase D proteins (61). The PX domains of these family members range in size, with highest homology in a region spanning ca. 60 amino acids. PX domains commonly contain a short proline-rich region, although it is not found in Vam7p. Interestingly, PX domains are also prominent in proteins implicated in vesicular trafficking, including the mammalian protein SNX1, which has been shown to interact with the cytosolic tail of the EGF receptor (47), and the yeast proteins Vps5p, Vps17p, Mvp1p, and Vam7p. The role of the PX domain has not been defined. However, it has been suggested to function in protein-protein interactions. Consistent with this idea, Vps5p and Vps17p have been shown to physically interact (37), although it is not known if their PX domains mediate this interaction. Synthetic interactions with the vam3^tsf mutation suggest that the PX domain of Vam7p may contribute to or regulate the interaction between these two proteins or it may mediate interactions with other components of the docking and fusion machinery. Further genetic and physical interaction studies will provide a greater understanding of PX domain function in Vam7p and possibly other PX domain-containing proteins.

Model for Vam7p as a SNAP-25-like molecule in the docking and fusion of transport intermediates to the vacuole. Based on the genetic and biochemical studies described here and an examination of protein transport in other systems, the specific roles for gene products involved in the late events of vacuolar protein sorting can be proposed (Fig. 8). Docking and/or fusion of prevacuolar transport intermediates from at least three distinct biosynthetic pathways to the vacuole requires the SNAP-25 family member Vam7p and the syntaxin homolog Vam3p. These interactions may be regulated by the Rab GTPase, Ypt7p (84, 86), and the VPS33 gene product, the SEC1 homolog shown to genetically interact with VAM3 (20). Lastly, transport to the vacuole may require Sec18p to activate and dissociate SNARE complexes, allowing for the docking and/or fusion of transport intermediates with the vacuole. Further investigation of components of the Vam3p SNARE complex will be required to understand the precise function of Vam7p. Screens to identify proteins that physically and genetically interact with the PX domain of Vam7p, such as the class C VPS genes that also function late in the pathway (65), will provide insight into the function of this domain and its involvement in docking and/or fusion mechanisms. Results from these examinations should help in understanding the molecular mechanisms that direct the specific docking and fusion of transport intermediates with the appropriate target membrane in both yeast and other eukaryotes.

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Model depicting genes involved in docking and/or fusion at the vacuole. Three vacuolar proteins proceed to the vacuole via distinct biosynthetic pathways, indicated above the arrows. Vam7p and Vam3p function as a t-SNARE complex in the docking and/or fusion of transport intermediates to the vacuole. Other genes that are proposed to regulate the docking and fusion of transport intermediates to the vacuole are enclosed by brackets. The homologies between these VPS genes and proteins implicated in vesicular docking and fusion are indicated (). The PX domain and coiled-coil domains of Vam7p and Vam3p are also schematically diagrammed.