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© The Rockefeller University Press, 0021-9525/1997//517 $5.00
The Journal of Cell Biology, Volume 138, Number 3, , 1997 517-529

A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole

Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668

Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3^tsf). Upon shifting the vam3^tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3^tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3^tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3 mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12 mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

Abbreviations used in this paper: ALP, alkaline phosphatase; API, aminopeptidase I; CPS, carboxypeptidase S; CPY, carboxypeptidase Y; ECL, enhanced chemiluminescence; FM4-64, [N-(3-triethylammoniumpropyl)- 4-(p-diethylaminophenylhexatrienyl) pyridinum dibromide]; GST, glutathione-S-transferase; ORF, open reading frame; PrA, proteinase A; YPD, yeast extract/peptone/dextrose.

IN eukaryotic cells, the functional properties of a particular intracellular organelle are largely defined by its unique set of resident proteins. Maintenance of organelle identity therefore requires effective sorting and delivery of proteins. Protein trafficking in the secretory pathway requires a series of events including cargo selection and vesicle budding at the donor organelle, followed by transport, docking, and fusion of transport vesicles with the appropriate target organelle.

Protein transport to the vacuole in the budding yeast Saccharomyces cerevisiae is one system in which these processes have been extensively studied. The yeast vacuole is an acidified compartment analogous to the lysosome of mammalian cells, containing a variety of hydrolytic enzymes that are responsible for macromolecular degradation (Klionsky et al., 1990). Resident vacuolar proteins and proteins destined for degradation are delivered to the vacuole via biosynthetic, endocytic, and autophagic transport routes. Thus, the vacuole represents a site of convergence for these distinct pathways. To identify the protein machinery involved in Golgi to vacuole protein transport, several mutant screens have been developed to detect vacuolar protein sorting (vps) (Bankaitis et al., 1986; Rothman and Stevens, 1986; Robinson et al., 1988; Rothman et al., 1989), vacuolar morphology (vam) (Wada et al., 1992), and vacuolar protease activity (pep) (Jones, 1977) mutants. Through these screens, >40 mutant complementation groups have been identified.

Yeast and mammalian cells appear to share a highly conserved machinery that functions in protein sorting and membrane fusion (Bennett and Scheller, 1993; Ferro-Novick and Jahn, 1994; Rothman, 1994). For example, members of the syntaxin, synaptobrevin/VAMP, Sec1p, and Rab protein families function at multiple stages of the secretory pathway (for reviews see Rothman, 1994; Pfeffer, 1996). Syntaxin (t-SNARE) and synaptobrevin (v-SNARE) family proteins are characterized as cytoplasmically oriented integral membrane proteins containing regions predicted to form coiled-coil domains (Bennett et al., 1993). A predominant model for the function of these proteins postulates that interactions between a particular syntaxin molecule (t-SNARE) localized on the target membrane with its corresponding VAMP/synaptobrevin molecule (v-SNARE) localized on the vesicle membrane provides the specificity required for faithful vesicle-mediated transport (Bennett and Scheller, 1993; Sollner et al., 1993). Members of the Sec1p and Rab families of proteins are thought to regulate the formation and/or activity of these SNARE complexes (for reviews see Novick and Brennwald, 1993; Rothman, 1994).

Several representatives of these conserved protein families have been identified in the vacuolar protein sorting pathway. For example, two distinct Rab proteins, Vps21p and Ypt7p (Wichmann et al., 1992; Horazdovsky et al., 1994; Singer-Kruger et al., 1994), and two distinct Sec1p homologues, Vps33p and Vps45p (Banta et al., 1990; Wada et al., 1990; Cowles et al., 1994; Piper et al., 1994), are required for Golgi to vacuole protein transport. These pairs of proteins define the two distinct stages in Golgi to vacuole protein transport. Pep12p, a syntaxin homologue, Vps45p, and Vps21p have been shown to direct Golgi to endosome protein trafficking (Becherer et al., 1996; Burd et al., 1997). Recently, Vam3p was identified as a syntaxin homologue associated with vacuolar membranes (Wada et al., 1997), suggesting that it may function together with Vps33p and Ypt7p in the endosome to vacuole transport reaction.

We describe experiments that test if Vam3p acts with other VPS gene products to direct endosome to vacuole transport. Both subcellular localization studies and analysis of a vam3^tsf mutant indicate that the primary function of Vam3p is at the vacuole where it accepts protein traffic from multiple transport pathways. Genetic studies indicate that Vam3p acts in conjunction with Vps33p, a Sec1p homologue, to execute its function. Finally, suppression studies show that Vam3p and Pep12p can partially substitute for one another, suggesting that SNARE molecules do not constitute the only specificity factor in vesicular targeting and fusion events.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Strains and Media
S. cerevisiae strains used for these studies are listed in Table I. Yeast strains were grown in standard yeast extract/peptone/dextrose (YPD),¹ yeast extract/peptone/fructose (YPF), or synthetic medium (YNB) containing 2% dextrose and supplemented as necessary with essential amino acids (Sherman et al., 1979). Standard bacterial medium (Miller, 1972) supplemented with 100 µg/ml ampicillin for plasmid retention was used to propagate Escherichia coli. Yeast strains used in EM examination of autophagy were grown in synthetic nitrogen starvation medium without amino acids and ammonium sulfate (SD-N) (Takeshige et al., 1992). Transformation of S. cerevisiae was done by the lithium acetate method (Ito et al., 1983). E. coli transformations were done as described previously (Hanahan, 1983).

Metabolic Labeling and Immunoprecipitation
To examine the biosynthetic transport of vacuolar proteins, cells were grown at 26°C in synthetic medium supplemented with amino acids to an OD₆₀₀ of 0.5–1.0. Cells were harvested and converted to spheroplasts as described previously (Paravicini et al., 1992). Spheroplasts were resuspended at a concentration of 3 OD₆₀₀/ml in synthetic medium containing amino acids and supplemented with 100 µg/ml 2-macroglobulin and 1 µg/ml BSA to stabilize secreted proteins. Cultures were preincubated at the appropriate experimental temperature for 5 min, and then labeled with 60 µCi [³⁵S]cysteine/methionine per ml of cell suspension. After labeling, cultures were chased with the addition of methionine, cysteine, yeast extract, and glucose to final concentrations of 5 mM, 1 mM, 0.4%, and 0.2%, respectively. After appropriate chase periods, samples were harvested into an equal volume of 2x energy poison buffer (40 mM sodium azide, 40 mM sodium fluoride, 50 mM Tris, pH 7.5, and 1 M sorbitol) and stored on ice for 2 min. The spheroplasts were then spun at 13,000 g for 2 min and separated into intracellular and extracellular fractions. The resulting samples were precipitated by the addition of TCA to a final concentration of 10%. For analysis of carboxypeptidase Y (CPY) sorting in vps33-8 cells, spheroplasting was performed after labeling and chase. After the desired chase period, the cultures were harvested into 2x spheroplast buffer (40 mM sodium azide, 40 mM sodium fluoride, 50 mM Tris, pH 7.5, and 2 M sorbitol, 20 mM DTT) and held on ice for 10 min. Zymolase was added to a concentration of 5 µg/OD₆₀₀ and the cells were incubated at 30°C for 30 min. The resulting spheroplasts were spun at 13,000 g for 2 min and separated into I and E fractions. Proteins were precipitated by the addition of TCA to a final concentration of 10%. Analysis of aminopeptidase I (API) and CPY performed in whole cells was done as previously described (Gaynor et al., 1994). Proteins were immunoprecipitated with specific antibodies as previously described (Rieder et al., 1996). Antibodies to API were a generous gift from Dan Klionsky (University of California, Davis) (Klionsky et al., 1992). Antibodies to CPY, proteinase A (PrA), alkaline phosphatase (ALP), carboxypeptidase S (CPS), Pep12p, and Vps10p have been previously characterized (Klionsky et al., 1988; Klionsky and Emr, 1989; Marcusson et al., 1994; Becherer et al., 1996; Cowles et al., 1997).

Preparation of Antisera against Vam3p
A hybrid protein fusing the amino-terminal 202 amino acids of Vam3p to glutathione-S-transferase (GST) was expressed in E. coli XL1Blue [supE44 thi-1 lac endA1 gyrA96 hsdR17 relA1 F'proAB lacI^qZM15]. GST–Vam3p fusion protein was isolated by affinity binding to glutathione-coupled Sepharose and further purified by SDS-PAGE. Purified protein was used to immunize New Zealand White rabbits as previously described (Horazdovsky and Emr, 1993). The crude antiserum was affinity purified by binding to cyanogen bromide–coupled GST–Vam3p (Harlow and Lane, 1988). Vam3p was detected in yeast cell lysates by immunoblotting and enhanced chemiluminescence (ECL) detection as previously described (Babst et al., 1997).

Subcellular Fractionation and Gradient Analysis
For intracellular localization of Vam3p, spheroplasts were made from wild-type cells and lysed in hypoosmotic lysis buffer as previously described (Gaynor et al., 1994). The crude lysate was centrifuged at 300 g to remove any unlysed spheroplasts. The 300 g supernatant was sequentially centrifuged at 13,000 g (15 min) and 100,000 g (60 min) to generate both low and high speed pellet and supernatant fractions. Resulting samples were precipitated in 10% TCA and analyzed by immunoblotting and ECL detection as described previously (Babst et al., 1997). Gradient Accudenz (Accurate Chemical and Scientific Corp., Westbury, NY) solutions were prepared (wt/vol) in hypoosmotic lysis buffer. The gradient was generated using the following Accudenz concentration steps from bottom to top: 0.5 ml 60%, 1 ml 50%, 1 ml 43%, 1 ml 37%, 1 ml 31%, 1 ml 27%, 1 ml 23%, 1 ml 20%, 1 ml 17%, 1 ml 13%, and 1 ml 7%. Gradient analysis was performed on 15 OD₆₀₀ equivalents of spheroplast lysate (300 g supernatant fraction) in a vol of 3 ml loaded on top of the gradient. The gradient was subjected to centrifugation at 4°C in an SW41 rotor (Beckman Instruments, Inc., Fullerton, CA) at 170,000 g for 18 h. 12 fractions were harvested manually from the top of the gradient, and proteins were TCA precipitated and analyzed by immunoblotting. mAbs to ALP were purchased from Molecular Probes, Inc. (Eugene, OR). Quantitation of proteins on gels was done by densitometry using NIH Image.

Construction of VAM3 and VPS33 Mutant Alleles
A temperature-conditional allele of VAM3 was constructed by PCR-based mutagenesis (Muhlrad et al., 1992). Primers complementary to chromosomal sequences immediately adjacent to the start and stop codons of VAM3 were used to amplify a 900-bp fragment (containing the entirety of the VAM3 ORF) under limiting dATP conditions (20 µM). A gapped plasmid was generated by digesting pVAM3.414 with BsmI and by isolating the vector by gel purification. The mutagenized PCR product and gapped plasmid were cotransformed into CJY1 cells, and transformants in which homologous recombination resulted in integration of mutagenized PCR product were selected by amino acid prototrophy. In much the same manner, a temperature-conditional allele of VPS33 was also constructed. Primers complementary to the sequence including both the start and stop codons of VPS33 were used to amplify a 2.1-kb PCR fragment under limiting dATP (20 µM) concentrations. Gapped plasmid was generated by digesting pVPS33.415 with AocI and SmaI and by isolating the vector by gel purification. The resulting PCR product was cotransformed with gapped plasmid into LBY317 harboring the pCYI50 plasmid, and transformants were selected for amino acid prototrophy. In both mutant selections, transformants were replica plated onto YPF, grown at 26°C overnight, and tested by colorimetric invertase assay (Horazdovsky et al., 1994) for temperature-conditional vps phenotype (CPY-invertase secretion) at 26°C and after 6 h of temperature shift to 38°C. Presumptive conditional mutant colonies were picked and retested, and plasmid linkage of the missorting phenotype was confirmed by retransformation of isolated plasmids into either TDY1 or LBY317 cells, respectively.

Fluorescence Microscopy and EM
To examine vacuolar morphology, [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridium dibromide] (FM4-64) labeling of yeast cell cultures was done as previously described (Vida and Emr, 1995), except the labeling was done at a concentration of 32 µM FM4-64 (Molecular Probes, Inc.) in YPD for 15 min at 30°C. For vacuolar inheritance assays, the time of labeling was increased to 45 min at 26°C and then cultures were chased for an additional hour at 26°C. Labeled cells were harvested, resuspended in fresh medium, and split into two equal aliquots that were further incubated at either 26°C or 38°C for 2 h. For conventional EM morphology studies, cells were grown at 26°C to mid-log phase. A portion of the cells were shifted to a nonpermissive temperature of 38°C for either 1 or 3 h. The cultures were processed for EM as previously described (Rieder et al., 1996). For the examination of autophagy by EM, cells were grown in YNB at 26°C to mid-log phase, harvested, washed once in SD-N medium, and resuspended at 1 OD₆₀₀ per ml in SD-N medium to induce autophagy (Takeshige et al., 1992). The cultures were split and shifted to either 26°C or 38°C for 2.5 h and then processed for EM.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Vam3p Is a Membrane-bound Protein That Localizes to the Vacuole
The sequence of Vam3p predicts a protein of 283 amino acids that, like other SNARE family members, has a transmembrane domain predicted at the carboxy terminus of the protein. To identify the VAM3 gene product, polyclonal antiserum was raised against a fusion protein consisting of the amino terminal 202 amino acids of Vam3p fused to GST. The resulting antiserum was affinity purified by binding to a GST–Vam3p-coupled cyanogen bromide column (Harlow and Lane, 1988). The affinity-purified antisera recognized a 35-kD protein from wild-type cell extracts by immunoblotting (Fig. 1 A, lane 2). The 35-kD species was not recognized by preimmune sera (data not shown) and was not present in vam3 cells (Fig. 1 A, lane 1). Cells expressing VAM3 from a 2µ overexpression plasmid showed a 15–20-fold increase in the abundance of the protein (Fig. 1 A, lane 3). The molecular mass of the protein was consistent with the 32-kD predicted molecular mass of Vam3p. Vam3p protein levels remained stable during a 60-min chase in cells treated with 20 µg/ml cycloheximide to inhibit new protein synthesis (data not shown). The relative abundance of Vam3p was 20-fold less than CPY (>0.05% of cell protein), indicating that Vam3p is a fairly rare protein (data not shown).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Localization of the VAM3 gene product. (A) SEY6210 (WT), TDY1 (vam3), and SEY6210 cells harboring VAM3 on a multicopy (2µ) plasmid (pVAM3.424) were grown to exponential phase, harvested by centrifugation, and lysed. Total cellular proteins (0.5 OD600 equivalent per lane in lanes 1 and 2, and 0.1 OD600 equivalent in lane 3) were separated by SDS-PAGE and transferred to nitrocellulose. Vam3p was immunoblotted with affinity-purified polyclonal antibody and visualized by ECL fluorography. Note that the exposure time of lane 3 is approximately half that of lanes 1 and 2. (B) Wild-type (SEY6210) cells were labeled with [35S]cysteine/methionine at 30°C for 15 min and chased for an additional 45 min. Lysed spheroplasts were fractionated by differential centrifugation (as described in Materials and Methods) generating P13, P100, and S100 fractions. The fractions were divided into two aliquots. ALP and Vps10p were immunoprecipitated from the first set of fractions, resolved by SDS-PAGE, and visualized by autoradiography. Total proteins (0.5 OD600 equivalents per fraction) from the second set of fractions were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with specific antiserum to Vam3p or Pep12p. Immunoblotted proteins were visualized by ECL fluorography. (C) A cleared lysate was generated from wild-type (SEY6210) cells, loaded at the top of an Accudenz step gradient, and centrifuged to equilibrium. Fractions were collected starting at the top of the gradient. Total proteins were precipitated from the fractions, separated by SDS-PAGE, and transferred to nitrocellulose. Vam3p, ALP, and Pep12p were detected by immunoblotting and visualized by ECL fluorography. The blot displaying the distribution of Vam3p is shown, and the distributions of Vam3p, ALP, and Pep12p are shown graphically.

Further support for vacuolar localization of Vam3p was provided by fractionation of cell lysates on an Accudenz equilibrium density gradient. Spheroplasts were prepared from wild-type cells and lysed as described above. The cleared lysate (300 g supernatant fraction) was loaded at the top of an Accudenz step gradient and centrifuged to equilibrium. Fractions were collected starting at the top of the gradient and analyzed for the presence of Vam3p, Pep12p, and ALP (Fig. 1 C). Vam3p colocalized with the majority of the vacuolar membrane protein ALP in the least dense region of the gradient (fractions 1–4). In contrast, the endosomal protein Pep12p migrated to a region of greater density (fractions 5–8) that was distinct from both the vacuolar marker ALP and Vam3p. In these gradients, protein markers of the Golgi, ER, mitochondria, and plasma membrane typically migrate into more dense regions of the gradient, also distinct from Vam3p and ALP (Singer-Kruger et al., 1993). Taken together, these data provide strong evidence that Vam3p is localized to vacuolar membranes.

VAM3 Null Mutants Exhibit Severe Vacuolar Protein Sorting and Morphology Defects
To determine the phenotypic consequences resulting from loss of Vam3p, deletion strains were constructed. Deletion constructs were generated by replacing 75% of the VAM3 open reading frame with either the HIS3 or LEU2 genes (see Materials and Methods). Cells deleted for VAM3 were viable and grew at rates comparable to those of wild-type cells at elevated temperatures (38°C), indicating that VAM3 is not required for growth, even at elevated temperatures. Examination of vam3 cells by both FM4-64 vital staining and EM revealed that these mutants completely lack normal vacuolar structures. Instead, numerous electron-transparent compartments were present in almost all cells (data not shown). In addition, many cells contained two to five vacuole-like electron-dense structures, but these organelles were significantly smaller than wild-type vacuoles (see Figs. 6 and 9). Accumulation of these novel electron-transparent membranous structures is characteristic of a subset of class B vps mutants, including vam3 (Wada et al., 1997) and vps41 (Cowles et al., 1997; Radisky et al., 1997), but not vps5 and vps17 (Kohrer and Emr, 1993; Horazdovsky et al., 1997).

View larger version (97K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Ultrastructural analysis of vam3tsf mutants. (A) A cross-section of a typical vam3tsf (TDY1 + pVAM3-6.414) cell grown at 26°C, which closely resembles wild-type cells. (B) A cross-section of a vam3tsf cell after temperature shift to 38°C for 3 h. The accumulation of novel compartments is seen in addition to a prominent, electron-dense vacuolar compartment. (C) Enlarged examples of structures seen in vam3tsf cells grown at 38°C for 3 h. (Arrows) Multivesicular bodies; (asterisk) electron-transparent membrane compartment. n, nucleus; v, vacuole. Bars: (A and B) 500 nm; (C) 200 nm.

View larger version (61K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Suppression of vam3 and pep12 mutant cell phenotypes by overexpression of the reciprocal t-SNARE. (A) TDY1 (vam3) cells containing either vector alone (pRS416), complementing plasmid (pVAM3.416), or PEP12 on a multicopy plasmid (pPEP12.426), and CBY31 (pep12) cells carrying vector only (pRS416), complementing plasmid (pPEP12.416), or multicopy VAM3 plasmid (pVAM3.426) were converted to spheroplasts. CCY120 (vps45) and CBY12 (vps45pep12) containing either vector (pRS424) or VAM3 on a multicopy plasmid (pVAM3.424) were harvested as whole cells. All strains were incubated for 5 min at 30°C and then labeled with [35S]cysteine/methionine for 10 min. Chase was initiated by the addition of unlabeled cysteine and methionine and incubation was continued for 30 min. CPY was immunoprecipitated from lysates, resolved by SDS-PAGE, and visualized by autoradiography. Golgi-modified precursor (p2) and mature (m) forms of CPY are indicated. (B) TDY1 (vam3) and CBY31 (pep12) transformed with the identical plasmids as in A were grown at 30°C to exponential phase, harvested, and labeled with FM4-64 for 30 min at 30°C. Chase was initiated by the addition of prewarmed YPD medium and incubation was continued at 30°C for 1 h. Cells were then viewed by fluorescence and Nomarski microscopy.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Vacuolar protein sorting in vam3 null cells. SEY6210 (WT), TDY1 (vam3), and TDY1 cells harboring single-copy complementing VAM3 plasmid (pVAM3.414) were converted to spheroplasts, and then pulse labeled with [35S]cysteine/methionine for 10 min at 30°C. Chase medium containing nonradioactive cysteine and methionine was added and incubation was continued for an additional 45 min. The spheroplasts were separated into intracellular (I) and extracellular (E) fractions, and CPY was immunoprecipitated with specific polyclonal antibodies, resolved by SDS-PAGE, and analyzed by autoradiography. The positions of Golgi-modified precursor (p2) and mature (m) CPY are indicated.

If Vam3p is directly required for vacuolar protein transport, then inactivation of the protein (i.e., by temperature shift) would be expected to result in an immediate block in vacuolar protein processing. We tested vam3^tsf cells for sorting of vacuolar hydrolases after a short incubation at nonpermissive temperature. Spheroplasts were prepared from both vam3^tsf and wild-type cells. The cultures were split and half was incubated at 26°C while the other half was shifted to 38°C for 5 min. Each culture was then pulse labeled for 10 min with [³⁵S]cysteine/methionine and chased with unlabeled cysteine/methionine for 45 min. Samples were harvested and separated into intracellular and extracellular fractions. Vacuolar proteins were immunoprecipitated with specific antibodies and analyzed by SDS-PAGE. As shown in Fig. 3, at the permissive temperature of 26°C, vam3^tsf cells matured CPY in a manner indistinguishable from wild-type cells (Fig. 3, lanes 1–4). However, at 38°C, a rapid block in the maturation of CPY was observed (Fig. 3, lanes 5 and 6). Approximately 60% of CPY accumulated as the Golgi-modified p2 precursor form. The remaining 40% of CPY was processed aberrantly and accumulated intracellularly. Less than 5% of the newly synthesized CPY was secreted to the extracellular media fraction. Similar results were observed for another soluble vacuolar hydrolase, PrA. We also examined the processing of two vacuolar membrane proteins, ALP and CPS. In vam3^tsf cells at permissive temperature, the processing of both ALP and CPS occurred in a manner identical to wild-type cells (Fig. 3, lanes 1–4). However, in vam3^tsf cells after a 5-min incubation at nonpermissive temperature, both proteins were completely blocked as the Golgi-modified precursor forms (Fig. 3, lanes 5 and 6). Vam3p thus appears to play an essential role in vacuolar protein transport of both soluble and integral membrane proteins.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Vacuolar protein sorting in vam3tsf mutant cells. TDY1 (vam3) cells transformed with either complementing plasmid (pVAM3.414) or plasmid containing a temperature-sensitive for function (tsf) allele of vam3 (pVAM3-6.414) were converted to spheroplasts, and then incubated at either permissive (26°C) or nonpermissive (38°C) temperature for 5 min. Cultures were labeled with [35S]cysteine/methionine for 10 min, and then chased for an additional 45 min at the indicated temperature. The cultures were separated into intracellular (I) and extracellular (E) fractions, and the vacuolar proteins CPY, PrA, CPS, and ALP were immunoprecipitated from each fraction, resolved by SDS-PAGE, and followed by autoradiography. CPS samples were treated with endoglycosidase H before electrophoresis. The positions of Golgi-modified precursor (p2, pro) and mature vacuolar (m) proteins are indicated.

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Analysis of API maturation in vam3tsf cells. Wild-type (SEY6210) cells and TDY1 (vam3) cells carrying a vam3tsf plasmid (pVAM3-6.414) were incubated at 38°C for 5 min, labeled with [35S]cysteine/methionine for 10 min, and then chased for the indicated times. Equivalent volumes of labeled culture were harvested at the indicated time points of chase. API was recovered from lysates by immunoprecipitation, subjected to SDS-PAGE, and analyzed by autoradiography. The cytoplasmic precursor (pr) and mature vacuolar (m) forms of API are indicated.

View larger version (67K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Examination of autophagy by EM. (A) Example of a pep4 cell (TDY10 harboring complementing VAM3 plasmid, pVAM3.414) after a 2.5-h induction of autophagy by nitrogen starvation at 38°C. (Arrowheads) The accumulation of autophagic bodies within the vacuole. (B) Cross-section of a vam3tsfpep4 cell (TDY10 cells harboring vam3tsf plasmid, pVAM3-6.414) after a 2.5-h induction of autophagy at a nonpermissive temperature of 38°C. An accumulation of autophagosomes in the cytoplasm (asterisks) is enclosed by the dashed box. (C) A region of the cell in B is enlarged in C, and arrows point to the membrane surrounding the autophagosomes. Fragments of the fragile limiting membrane are visible (arrows). n, nucleus; v, vacuole. Bars: (A and B) 500 nm; (C) 200 nm.

Vacuolar Inheritance Is Unaffected in vam3^tsf Cells
Since multiple fusion events with the vacuole are disrupted because of the inactivation of Vam3p, we were interested in investigating the effect of Vam3p on vacuolar inheritance, a process thought to involve homotypic vacuole–vacuole fusion (Conradt et al., 1992). Therefore, we examined the ability of vam3^tsf cells to properly segregate vacuoles into the daughter bud. The vacuoles of both wild-type and vam3^tsf cells were labeled with FM4-64 for 45 min at 26°C. The cells then were chased in YPD for 1 h at 26°C and split into two aliquots. One aliquot was maintained at 26°C and the other was shifted to 38°C for 2 h. Cells were harvested and viewed by fluorescence microscopy for the presence of vacuolar segregation structures. At either 26°C or 38°C, vam3^tsf cells formed segregation structures in a manner indistinguishable from wild type; in both cases at least 96% of budding cells (n = 200 cells) had vacuoles in both the mother and daughter cells (Fig. 7). Furthermore, vam3^tsf cells, even after 2 h at nonpermissive temperature, extended continuous segregation structures into the daughter bud and maintained intact vacuoles. Therefore, the function of Vam3p does not appear to be required for proper vacuolar segregation during mitosis.

View larger version (104K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Vacuolar inheritance analysis of vam3tsf cells. TDY1 (vam3) cells carrying either complementing VAM3 plasmid (pVAM3.414) or vam3tsf plasmid (pVAM3-6.414) were grown to exponential phase and then labeled with 32 µM FM4-64 for a period of 45 min. Cultures were chased with excess YPD for 1 h at 26°C and then split into equal aliquots. The aliquots were chased for an additional 2 h at either 26°C or 38°C. The cultures were then examined by fluorescence and Nomarski microscopy for the presence of vacuolar segregation structures. (White arrowheads) Segregation structures.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 8 vps33tsfvam3tsf double mutant cells display synthetic vacuolar protein sorting defects. (A) LBY317 (vps33) cells harboring complementing VPS33 plasmid (pVPS33.415) or vps33tsf plasmid (VPS33-8.415) were incubated at either 26°C or 38°C for 5 min, and then labeled with [35S]cysteine/methionine for 10 min. Chase was initiated by the addition of nonradioactive cysteine and methionine and incubation was continued for 30 min. Cells were spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY was immunoprecipitated from each fraction, resolved by SDS-PAGE, and viewed by autoradiography. (B) vam3tsf (TDY7 + pVPS33.416 and pVAM3-6.414) and vps33tsf (pTDY7 + pVAM3.414 and pVPS33-8.416) single mutant cells, and vam3tsfvps33tsf (TDY7 + pVAM3-6.414 and pVPS33-8.416) double mutant cells were incubated at 26°C for 5 min, labeled with [35S]cysteine/methionine for 10 min, and chased for 30 min. CPY was recovered by immunoprecipitation, separated by SDS-PAGE, and followed by autoradiography. The positions of both Golgi-modified precursor (p2) and mature (m) CPY are indicated.

Pep12p and Vam3p Can Partially Substitute for One Another in the Vacuolar Protein Transport Pathway
Although Vam3p shares significant homology with many members of the syntaxin family, it is most similar to Pep12p, an endosomal t-SNARE. To address whether these SNARE proteins can functionally substitute for one another, we overexpressed each SNARE in the reciprocal null mutant background and examined the strains for improved transport of CPY to the vacuole. Spheroplasts were prepared from each strain, radiolabeled, and chased; proteins were then immunoprecipitated and analyzed by SDS-PAGE (Fig. 9 A). In both vam3 and pep12 cells, CPY accumulated as the Golgi-modified precursor form (Fig. 9 A, lanes 1 and 4). Introduction of CEN plasmids harboring VAM3 or PEP12 into vam3 or pep12 cells, respectively, complemented the CPY sorting defects of these mutants (Fig. 9 A, lanes 3 and 6). Interestingly, the overexpression of Pep12p in vam3 cells and Vam3p in pep12 cells resulted in significant conversion of CPY to the mature vacuolar form (Fig. 9 A, lanes 2 and 5). In addition to suppression of the CPY sorting defect, the ALP maturation defects were similarly suppressed (data not shown). To further determine the requirements of VAM3 suppression of pep12 cells, we examined the effect of VAM3 overexpression in vps45 and vps45pep12 cells. VPS45 gene encodes a member of the Sec1p family of proteins (Cowles et al., 1994; Piper et al., 1994) and is required, with Pep12p, for Golgi to endosome protein transport (Burd et al., 1997). Cultures of each strain were radiolabeled and chased, and proteins were immunoprecipitated and resolved by SDS-PAGE (Fig. 9 A). In both vps45 mutant cells and vps45pep12 double mutant cells, CPY was blocked in the Golgi-modified precursor form (Fig. 9 A, lanes 7 and 8). Overexpression of VAM3 was unable to suppress the CPY sorting defects of either mutant strain (Fig. 9 A, lanes 8 and 9). These results indicate that VAM3 requires VPS45 to facilitate CPY transport in pep12 cells. In a reciprocal experiment we found that overexpression of PEP12 does not suppress a vps33 mutant (data not shown).

Suppression of morphological defects associated with vam3 and pep12 mutant cells was also examined by vital staining of the vacuoles with FM4-64. As shown in Fig. 9 B, wild-type cells typically contain several brightly staining vacuolar structures that corresponded to vacuolar depressions in Nomarski images (Vida and Emr, 1995). In contrast, vam3 cells displayed a punctate staining pattern throughout the cytoplasm. pep12 cells stained with FM4-64 displayed a single, large, vacuolar structure, characteristic of class D vps mutants (Vida and Emr, 1995). Overexpression of Pep12p dramatically suppressed the morphological defects of vam3 cells, resulting in several normal appearing vacuole structures. Similarly, pep12 cells that overproduced Vam3p contained near wild-type vacuolar structures. The introduction of VAM3 or PEP12 on low copy plasmids completely complemented the morphological defects of both the vam3 cells and pep12 cells, respectively. Overexpression of other presumptive yeast SNARE molecules (Sec22p, Bos1p, ORF #D8035.11p, ORF #YLR0936) or other class D proteins (Vps45p, Vac1p, Vps9p, Vps8p) did not improve vacuolar protein sorting of vam3 cells, indicating that the suppression by Pep12p is specific.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We report on the primary function of Vam3p, a syntaxin homologue, in the docking and/or fusion of transport intermediates from multiple protein sorting pathways to the vacuole/lysosome. Subcellular fractionation and density gradient data indicated that Vam3p colocalized with vacuolar membranes. Conditional vam3^tsf mutants exhibited a rapid defect in the transport of multiple resident vacuolar proteins after shifting to the nonpermissive temperature, indicating that Vam3p is a component of the vacuolar protein transport machinery. Additionally, autophagic pathway intermediates and API precursor accumulated in vam3^tsf cells, indicating that Vam3p is also required for autophagy. Finally, EM analysis of these conditional mutants revealed a temperature-dependent accumulation of novel membrane structures, including multivesicular bodies, which may represent distinct transport intermediates destined for fusion with the vacuole. Thus, our data are most consistent with Vam3p residing on the vacuolar membrane where it functions as a multispecificity receptor required for docking and/or fusion of multiple prevacuolar transport intermediates.

Members of the syntaxin family of proteins are thought to act at specific intracellular organelles as receptors for the selective docking of appropriate transport intermediates. For instance, in neuronal cells, syntaxin is localized to the plasma membrane where it mediates the docking and fusion of secretory vesicles containing neurotransmitters (Bennett et al., 1992, 1993). In yeast, Sso1p and Sso2p, two redundant syntaxin homologues whose function is required for secretion, are localized to the plasma membrane (Aalto et al., 1993; Brennwald et al., 1994). Furthermore, Pep12p, an endosomal protein, is required for Golgi to endosome protein transport (Becherer et al., 1996; Burd et al., 1997). Thus, the membrane localization of such proteins is suggestive of their site of function. We have demonstrated through subcellular localization experiments that the majority of Vam3p resides in vacuolar membranes. It has also been reported recently that Vam3p is localized to vacuolar membranes by indirect immunofluorescence (Wada et al., 1997). Together, these data provide strong evidence that Vam3p resides on the vacuole, consistent with its function as a vacuolar t-SNARE.

Vam3p Functions as a Multispecificity t-SNARE
Our analysis of a temperature-conditional vam3 mutant (vam3^tsf) suggests a direct involvement of Vam3p in a late step of protein transport to the vacuole. Multiple transport pathways to the vacuole were blocked even after a short incubation of vam3^tsf cells at nonpermissive temperature. There are at least two pathways of biosynthetic traffic to the vacuole. CPY, PrA, and CPS transit through an endosomal compartment before final delivery to the vacuole. ALP is delivered by an alternate Golgi-to-vacuole transport pathway, possibly bypassing the endosomal compartment (Cowles et al., 1994, 1997; Stack et al., 1995; Burd et al., 1996, 1997; Horazdovsky et al., 1996). vam3^tsf cells displayed immediate defects in the transport of CPY, PrA, CPS, and ALP, which is indicative of a direct involvement of Vam3p in both biosynthetic transport pathways.

Interestingly, the fate of accumulated p2CPY in vam3^tsf cells suggests that Vam3p acts at a late step in the vacuolar protein sorting pathway. Defects in receptor-mediated transport of CPY from the Golgi to the endosome result in the rapid secretion of CPY (Cowles et al., 1994; Piper et al., 1994; Stack et al., 1995; Burd et al., 1997). Unlike these early acting conditional vps mutants (pep12^tsf and vps45^tsf mutants) that secrete p2CPY immediately upon inactivation, vam3^tsf cells accumulated p2CPY intracellularly. The lack of CPY secretion in vam3^tsf cells at nonpermissive temperature indicates that transport and recycling of the Vps10p hydrolase sorting receptor between the Golgi and the prevacuolar endosome is relatively unaffected and is thus consistent with a block in transport at a point beyond the endosomal intermediate. Additionally, the accumulated intracellular p2CPY in vam3^tsf cells at nonpermissive temperature is slowly processed to aberrant forms. This processing is unlikely to be occurring in the vacuole. These cells have normal vacuoles populated with active processing proteases that would rapidly process p2CPY to its mature form. Similar aberrant processing of vacuolar protein precursors has been observed in other vps mutants that have defects in endosome to vacuole transport as a result of accumulation in a proteolytically competent endosomal compartment (Babst et al., 1997). Extended accumulation of CPY in a late prevacuolar compartment in vam3^tsf cells results in slow maturation of p2CPY (t_1/2 of 60 min). Consistent with these observations, at nonpermissive temperature, vam3^tsf cells accumulated novel membrane-enclosed compartments that may represent these blocked transport intermediates.

The maturation of API, a protein delivered to the vacuole directly from the cytosol, possibly by an autophagic pathway (Klionsky et al., 1992), is also completely blocked at nonpermissive temperature in vam3^tsf cells. Furthermore, the docking/fusion of large autophagosomes with the vacuole is disrupted in vam3^tsf cells at nonpermissive temperature. Thus, the delivery of autophagic intermediates appears to require a functional vacuolar t-SNARE. If the mechanism of autophagic vesicle fusion uses conserved components such as SNAREs, autophagosomes may contain complementary components on their membranes that specifically target them to the vacuole via interactions with Vam3p.

The vam3 mutant was originally identified as a vacuolar morphology mutant (Wada et al., 1992). This phenotype is consistent with a primary role for Vam3p in maintenance of vacuolar morphology. However, in vam3^tsf cells, the vacuoles remain stable for >3 h at nonpermissive temperature. Instead, vam3^tsf cells accumulate novel membrane structures, which suggests that the primary defect associated with the loss of Vam3p function is impaired docking and/or fusion of transport intermediates with the vacuole. Thus, the complex morphology of vam3 cells appears to result from the accumulation of these transport intermediates, rather than instability and fragmentation of the vacuoles. Recent in vitro studies analyzing extracts from vam3 cells indicated that Vam3p is involved in homotypic vacuole–vacuole fusion (Nichols et al., 1997). While our data do not rule out a role for Vam3p in homotypic fusion, they suggest that these in vitro observations may correspond to heterotypic fusion of isolated prevacuolar intermediates from vam3 cells with wild-type vacuoles. Moreover, because we observed no obvious vacuolar inheritance defects in vam3^tsf cells incubated for extended time at nonpermissive temperature, Vam3p function does not appear to play an essential role in vacuole–vacuole fusion during the inheritance process. Similar in vitro fusion experiments analyzing vacuoles isolated from vam3^tsf cells may offer a means to determine the role, if any, of Vam3p in homotypic vacuole fusion reactions.

The accumulation of protein precursors and membrane-bound intermediate compartments that transit to the vacuole via distinct routes is consistent with a transport block at a site where these various pathways converge, the vacuole (Fig. 10). While we cannot rule out the possibility that Vam3p mediates the delivery of an unstable component, which in turn is required for the docking and fusion of other transport intermediates, this explanation seems unlikely because other vps mutants (i.e., pep12) do not show similar general transport defects (Burd et al., 1997; Cowles et al., 1997). Thus, analysis of protein transport and vacuolar morphology in vam3^tsf cells is consistent with Vam3p acting as a multispecificity vacuolar t-SNARE that mediates the docking/fusion of multiple distinct late transport intermediates with the vacuole. The early Golgi t-SNARE, Sed5p, may also function as a multispecificity t-SNARE as it has been shown to function in both biosynthetic ER-to-Golgi and retrograde late-to-early Golgi transport (Hardwick and Pelham, 1992; Banfield et al., 1994, 1995).

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Vam3p acts as a receptor at the vacuole for the docking and fusion of multiple transport intermediates. Pep12p, the endosomal t-SNARE, functions together with Sec1p family member Vps45p in Golgi to endosome transport of p2CPY as well as other soluble vacuolar hydrolases. Our analyses of Vam3p indicate that it functions on the vacuole together with Vps33p in the docking and fusion of multiple transport intermediates. Proteins that are targeted to the vacuole through two separate biosynthetic pathways, as well as those that are targeted to the vacuole directly from the cytoplasm by autophagy, are shown. In the absence of functional Vam3p at the vacuole, the various transport intermediates are unable to fuse with the vacuole and accumulate as novel membrane compartments in the cytoplasm.

Some insight into the function of syntaxin and Sec1p homologues can be gained by our observations that overexpression of Vam3p partially suppresses both the vacuolar protein sorting and morphological defects of a pep12 strain and, conversely, that overexpression of Pep12p results in partial suppression of the sorting and morphological defects associated with a vam3 strain. Apparently, increased cellular levels of these t-SNAREs results in redistribution of the excess protein to compartments other than their normal resident organelles, thus allowing them to function at inappropriate compartments. Indeed, overexpression of Pep12p results in a substantial pool of the protein redistributing to the vacuolar membrane (data not shown). Interestingly, overexpression of VAM3 and PEP12 was not capable of suppressing deletions of VPS45 and VPS33, respectively. Therefore, it is possible that under these conditions Pep12p and Vam3p may transiently interact with inappropriate components of the docking/fusion machinery to execute their function (i.e., Vam3p with Vps45p, and Pep12p with Vps33p). These observations indicate that SNARE molecules may not be the only specificity factors necessary for protein targeting. In fact, other studies indicate additional factors are required to direct docking and fusion of transport intermediates. For example, in mammalian cells, syntaxin is ubiquitously distributed throughout the neuronal plasma membrane, yet fusion occurs primarily at the active site of the nerve terminal, indicating that other factors are acting to direct fusion to this specific region (Bennett et al., 1992; Sollner et al., 1993; Garcia et al., 1995). Several candidate proteins that may be involved in this additional specificity function include members of the Sec1 and Rab protein families.

Studies examining the physical and genetic relationships between Vam3p and other transport components, as well as in vitro reconstitution of protein transport to the vacuole, are necessary to determine the mechanistic details of this final step of protein trafficking. Through these types of investigations, significant progress will be made in elucidating the molecular mechanisms of protein and membrane transport in yeast and other eukaryotic organisms.

	Acknowledgments

 
This work was supported by grants GM32703 and CA58689 from the National Institutes of Health to S.D. Emr. S.D. Emr is an investigator of the Howard Hughes Medical Institute.

Submitted: 28 April 1997

Revised: 9 June 1997

We are very grateful to Michael McCaffery and Tammie McQuistan for outstanding EM work (Electron Microscopy Core B, Program Project grant CA58689). We thank Yoh Wada and Dan Klionsky for helpful discussions and for generously providing plasmids and antisera. We also thank Colin Jamora for assistance with initial experiments, and members of the Emr laboratory, especially Erin Gaynor, Marcus Babst, and Chris Burd, for helpful comments and for critical reading of this manuscript.

Please address all correspondence to Scott D. Emr, Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, CA 92093-0668. Tel.: (619) 534-6462. Fax: (619) 534-6414.

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