DNA Methods

Recombinant DNA manipulations were performed using standard methods (Maniatis et al., 1982). Restriction and modification enzymes were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) and New England Biolabs (Beverly, MA). A YEp13-based genomic library plasmid (p351R1) containing the VAM3 open reading frame (ORF) and a vam3::HIS3 deletion construct were a generous gift from Yoh Wada (University of Tokyo, Japan) (Wada et al., 1997). Plasmids pVAM3.BS, pVAM3.414, pVAM3.424, and pVAM3.416 were generated by subcloning the 2.4-kb BstBI–NsiI fragment (containing the entire VAM3 coding sequence) of p351R1 into pBluescript KS(−) (Stratagene, La Jolla, CA), pRS414, pRS424, and pRS416 (Sikorski and Hieter, 1989), respectively. A VAM3 deletion construct was generated by replacing the BsmI fragment of pVAM3.BS (eliminating 75% of the VAM3 coding sequence) with the LEU2 gene. A linear DNA fragment containing the deletion construct was generated by PCR with primers complementary to the VAM3 sequence. Transformation of wild-type cells with the deletion construct resulted in homologous recombination and insertion of the auxotrophic marker at the chromosomal VAM3 locus. Transformants were selected by amino acid prototrophy and deletions were confirmed by PCR analysis of the chromosomal DNA. Plasmid pVAM3-6.414 containing a temperature-conditional allele of vam3 (vam3.6) was constructed by gapped plasmid repair (Muhlrad et al., 1992). The BamHI–KpnI fragment of pVAM3-6.414 containing vam3.6 was then subcloned into pRS416 (Sikorski and Hieter, 1989) to generate pVAM3-6.416. Plasmid pVPS33-8.415 containing a temperature-conditional allele of vps33 (vps33.8) was generated by gapped plasmid repair. The SalI–NotI fragment of pVPS33-8.415 containing the vps33.8 allele was subsequently subcloned into pRS416 (Sikorski and Hieter, 1989) to generate pVPS33-8.416. Plasmids pCYI50, pCB31, and pLB221 were described previously (Johnson et al., 1987; Banta et al., 1990; Burd et al., 1997).

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 α2-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 2× 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 2× 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^qZΔM15]. 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.

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 vam3Δ 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. ​Figs.66 and ​and9).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).

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Figure 6

Ultrastructural analysis of vam3^tsf mutants. (A) A cross-section of a typical vam3^tsf (TDY1 + pVAM3-6.414) cell grown at 26°C, which closely resembles wild-type cells. (B) A cross-section of a vam3^tsf 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 vam3^tsf 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.

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Figure 9

Suppression of vam3Δ and pep12Δ mutant cell phenotypes by overexpression of the reciprocal t-SNARE. (A) TDY1 (vam3Δ) cells containing either vector alone (pRS416), complementing plasmid (pVAM3.416), or PEP12 on a multicopy plasmid (pPEP12.426), and CBY31 (pep12Δ) cells carrying vector only (pRS416), complementing plasmid (pPEP12.416), or multicopy VAM3 plasmid (pVAM3.426) were converted to spheroplasts. CCY120 (vps45Δ) and CBY12 (vps45Δpep12Δ) 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 (vam3Δ) and CBY31 (pep12Δ) 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.

The abnormal vacuolar morphology of vam3Δ cells suggested that both protein transport to the vacuole and vacuolar function may be compromised in these cells. Therefore, the ability of vam3Δ cells to properly transport newly synthesized resident vacuolar proteins was examined. Spheroplasts prepared from vam3Δ cells were pulse labeled for 10 min with [³⁵S]cysteine/methionine to label newly synthesized proteins, and then were chased with unlabeled cysteine/methionine for 45 min. The spheroplasts were separated into intracellular and extracellular fractions. CPY was immunoprecipitated from the samples and analyzed by SDS-PAGE and autoradiography. The biosynthesis of vacuolar proteins such as CPY can be monitored by posttranslational modifications that correlate with transport through the secretory and vacuolar protein sorting pathways. In the case of CPY, p1CPY is generated as a result of core glycosyl modifications in the ER, which are then elongated in the Golgi complex to generate the slightly larger form, p2CPY. Upon delivery to the vacuole, the precursor CPY is cleaved to generate the mature active form of the enzyme, mCPY. As shown in Fig. ​Fig.2,2, wild-type cells properly delivered CPY to the vacuole as shown by the presence of mature CPY in the intracellular fraction (Fig. ​(Fig.2,2, lanes 1 and 2). In vam3Δ cells, CPY accumulated as the Golgi-modified p2 form, ~50% of which was secreted into the extracellular fraction (Fig. ​(Fig.2,2, lanes 3 and 4). A small portion (~10%) of the CPY that remained in the intracellular fraction was seen as an apparently misprocessed form that migrated as a species slightly smaller than p2CPY, which eventually was processed to mature CPY after prolonged chase (t_1/2 > 60 min). The introduction of VAM3 on a single copy, centromere (CEN)–based plasmid complemented the CPY maturation defect of the vam3Δ mutant cells (Fig. ​(Fig.2,2, lanes 5 and 6).

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Figure 2

Vacuolar protein sorting in vam3 null cells. SEY6210 (WT), TDY1 (vam3Δ), 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.

A vam3^tsf Mutant Is Defective for Transport of Multiple Vacuolar Proteins

Because of the dramatic effects on vacuolar morphology in vam3Δ cells, it is difficult to conclude whether the defects in protein sorting are a direct consequence of loss of Vam3p function, or due to secondary pleiotropic effects resulting from loss of vacuolar integrity in vam3Δ cells. To address the primary role of Vam3p, we generated a temperature-conditional allele of VAM3 by error prone PCR-mediated mutagenesis (Muhlrad et al., 1992; Stack et al., 1995). Approximately 6,000 transformants were screened for a vps phenotype (secretion of CPY–invertase fusion protein) at 26°C and after 6 h of temperature shift to 38°C. Several temperature-conditional alleles were obtained that secreted a substantial amount of CPY–invertase after a temperature shift to 38°C, but not at 26°C. One allele (vam3-6), which will be referred to here as vam3^tsf, was chosen and characterized further.

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. ​Fig.3,3, at the permissive temperature of 26°C, vam3^tsf cells matured CPY in a manner indistinguishable from wild-type cells (Fig. ​(Fig.3,3, lanes 1–4). However, at 38°C, a rapid block in the maturation of CPY was observed (Fig. ​(Fig.3,3, 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. ​(Fig.3,3, 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. ​(Fig.3,3, lanes 5 and 6). Vam3p thus appears to play an essential role in vacuolar protein transport of both soluble and integral membrane proteins.

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Figure 3

Vacuolar protein sorting in vam3^tsf mutant cells. TDY1 (vam3Δ) 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.

Vam3p Function Is Required for Cytoplasm to Vacuole Delivery of API

Some newly synthesized vacuolar proteins do not reach the vacuole via the secretory pathway but instead follow a direct cytosol to vacuole delivery pathway (Klionsky et al., 1992). The precursor form of API is synthesized in the cytoplasm and transported directly into the vacuole where the amino-terminal precursor is cleaved, producing the mature form of the enzyme (Klionsky et al., 1992). Many mutants defective for the processing of API (cvt mutants) are also defective in autophagy (aut and apg mutants), indicating that delivery of API to the vacuole may be mediated by an autophagic mechanism (i.e., macroautophagy) (Harding et al., 1996; Scott et al., 1996). The processing of API was examined in vam3^tsf cells to determine if Vam3p is required for delivery of API to the vacuole. Wild-type and vam3^tsf cells were shifted to nonpermissive temperature for 5 min and then labeled for 10 min. Samples were harvested after 0, 30, 60, 90, and 120 min of chase. The maturation of API was analyzed by immunoprecipitation, SDS-PAGE, and autoradiography. As shown in Fig. ​Fig.4,4, cells containing the wild-type copy of VAM3 matured API with normal kinetics; essentially all API was processed to the mature vacuolar form after 120 min of chase. However, vam3^tsf cells at nonpermissive temperature (38°C) accumulated newly synthesized API in its precursor form, with virtually no maturation of the protein, even after 120 min of chase. In vam3^tsf cells at the permissive temperature of 26°C, API was also matured with kinetics comparable to that of wild-type cells (data not shown). The defect in API processing observed in vam3^tsf cells at nonpermissive temperature indicates that Vam3p is directly required for the delivery of API to the vacuole.

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Figure 4

Analysis of API maturation in vam3^tsf cells. Wild-type (SEY6210) cells and TDY1 (vam3Δ) cells carrying a vam3^tsf 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.

Vam3p Is Required for the Docking and/or Fusion of Autophagosomes with the Vacuole

The block of API maturation in vam3^tsf cells suggested that targeting of autophagosomes to the vacuole may be disrupted. Autophagosomes appear by EM as membrane-enclosed structures between 400 and 600 nm in diam (Baba et al., 1994). The compartments are primarily composed of cytoplasmic material, as they direct bulk uptake and vacuolar transport of cytoplasmic constituents, and thus stain with an electron density similar to the cytoplasm (Baba et al., 1994). To further examine possible autophagic defects associated with the loss of Vam3p function, we induced the formation of autophagic intermediates by nitrogen starvation and examined their uptake by the vacuole in both wild-type and vam3^tsf cells. vam3^tsf cells and complemented vam3Δ cells, both of which were also deleted for the PEP4 gene to prevent vacuolar degradation of autophagic bodies, were grown at 26°C to mid-log phase in YNB medium. The cells were then harvested and resuspended in SD-N. The cultures were split and incubated at either 26°C or 38°C for 2.5 h. Cells were harvested, fixed, and processed for EM. Wild-type cells starved at both 26°C and 38°C, as well as vam3^tsf cells that were starved at 26°C, accumulated numerous autophagic bodies in the vacuoles (Fig. ​(Fig.55 A). In contrast, vam3^tsf cells starved at 38°C for 2.5 h accumulated multiple autophagosomes in the cytoplasm, with no detectable accumulation of autophagic bodies in the vacuole (Fig. ​(Fig.55 B). The double membranes surrounding the cytoplasmic autophagosomes appear to be very fragile and easily disrupted as a result of preparation for EM. Thus, the perimeter of the autophagosomes was often seen as the electron-transparent space that the membrane previously occupied (Fig. ​(Fig.55 C). The temperature-conditional block of autophagosome delivery to the vacuole suggests that this process is dependent on the function of Vam3p.

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Figure 5

Examination of autophagy by EM. (A) Example of a pep4Δ 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 vam3^tsfpep4Δ cell (TDY10 cells harboring vam3^tsf 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.

Morphological Analysis of vam3^tsf Cells Reveals Accumulation of Aberrant Membrane Compartments

Due to the dramatic accumulation of novel membrane compartments in vam3Δ cells, we also were interested in the onset, severity, and characteristics of morphological defects associated with the vam3^tsf allele. vam3^tsf cells that had been grown at permissive temperature (26°C) or shifted to nonpermissive temperature (38°C) for either 1 or 3 h were characterized by EM. At 26°C, vam3^tsf cells displayed typical wild-type morphology, containing prominent, intact, electron-dense vacuoles (Fig. ​(Fig.66 A). Although the majority of vam3^tsf cells that had been shifted to 38°C for both 1 and 3 h maintained intact vacuoles, an accumulation of 300–500-nm novel membrane-enclosed compartments was also observed (Fig. ​(Fig.66 B). The accumulation of these aberrant compartments increased with respect to time of incubation at 38°C. The compartments were heterogeneous in both shape and content as indicated by electron-dense or -transparent staining (Fig. ​(Fig.6,6, B and C). In addition, vam3^tsf cells at high temperature displayed an accumulation of multivesicular membrane compartments (Fig. ​(Fig.66 C). These data are consistent with a requirement for Vam3p in the docking/fusion of multiple transport intermediates with the vacuole.

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. ​(Fig.7).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.

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Figure 7

Vacuolar inheritance analysis of vam3^tsf cells. TDY1 (vam3Δ) cells carrying either complementing VAM3 plasmid (pVAM3.414) or vam3^tsf 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.

Vam3p Genetically Interacts with Vps33p, a Sec1p Homologue

Distinct protein complexes are thought to mediate vesicle targeting and fusion at discrete steps in the secretory and vacuolar protein sorting pathways. For example, members of the Sec1 family of proteins are thought to regulate vesicle targeting and fusion through specific interactions with t-SNARE proteins (Garcia et al., 1994; Pevsner et al., 1994). Thus, just as Pep12p has been shown to interact with the Sec1p homologue Vps45p to mediate protein sorting from the Golgi to the endosome (Burd et al., 1997), Vam3p is also likely to interact with regulatory proteins to execute its function. A strong candidate for such an interaction with Vam3p is Vps33p, a Sec1p homologue that may act at a late step in the vacuolar protein sorting pathway (Banta et al., 1990; Wada et al., 1990). Therefore, genetic interactions between VAM3 and VPS33 were examined. First, temperature-conditional alleles of VPS33 were generated by PCR-based mutagenesis in a manner similar to that described for VAM3 (see Materials and Methods). One temperature-conditional mutant (vps33-8), which will be referred here as vps33^tsf, was chosen for further characterization. After a 5-min incubation at either permissive (26°C) or nonpermissive (38°C) temperature, vps33^tsf cells were pulse labeled for 10 min and then chased for 30 min. After chase, the cells were spheroplasted and separated into intracellular and extracellular fractions. The processing of CPY was analyzed in each fraction by immunoprecipitation, SDS-PAGE, and autoradiography (Fig. ​(Fig.88 A). At 26°C, vps33^tsf cells properly processed the majority of CPY to the mature vacuolar form, with only a minor (~10%) portion delayed intracellularly in the p2 form (Fig. ​(Fig.88 A, lanes 5 and 6). However, in vps33^tsf cells after a 5-min incubation at 38°C, CPY was blocked intracellularly as the Golgi-modified p2 form of CPY. Similar to vam3^tsf cells, only a small percentage (<5%) of p2CPY was secreted into the extracellular fraction and a small portion of the p2CPY was misprocessed (Fig. ​(Fig.88 A, lanes 7 and 8). Wild-type cells properly matured CPY at both temperatures (Fig. ​(Fig.88 A, lanes 1–4). A defect in the processing of the vacuolar membrane protein ALP also was observed, with a subtle processing defect at the permissive temperature and a complete block in ALP maturation at the nonpermissive temperature (data not shown). Thus, like vam3^tsf cells, vps33^tsf mutants exhibited a rapid defect in the transport of both CPY and ALP to the vacuole at nonpermissive temperature.

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Figure 8

vps33^tsfvam3^tsf double mutant cells display synthetic vacuolar protein sorting defects. (A) LBY317 (vps33Δ) cells harboring complementing VPS33 plasmid (pVPS33.415) or vps33^tsf 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) vam3^tsf (TDY7 + pVPS33.416 and pVAM3-6.414) and vps33^{t sf} (pTDY7 + pVAM3.414 and pVPS33-8.416) single mutant cells, and vam3^tsfvps33^tsf (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.

To determine whether Vam3p and Vps33p might functionally interact, we examined a vam3^tsfvps33^tsf double mutant for synthetic vacuolar protein sorting defects. A haploid double mutant strain was constructed and CPY maturation was assayed by metabolic labeling of whole cells. Cells were pulse labeled for 10 min and then chased for an additional 30 min at 26°C. After chase, the cells were lysed, and proteins were immunoprecipitated, separated on SDS-PAGE, and visualized by autoradiography (Fig. ​(Fig.88 B). Under these conditions, both vam3^tsf and vps33^tsf single mutant cells matured >90% of CPY (Fig. ​(Fig.88 B, lanes 1 and 2). However, in vam3^tsfvps33^tsf double mutant cells, a moderate synthetic sorting defect was observed; ~50% of newly synthesized CPY remained blocked in the p2 precursor form (Fig. ​(Fig.88 B, lane 3). The synthetic sorting defect of the double mutant strain is not as severe as that of the single mutant strains at nonpermissive temperature, indicating that transport to the vacuole is not completely disrupted in the double mutant cells. However, the increased sorting defect suggests that the two proteins function together to mediate protein sorting to the vacuole.

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 vam3Δ 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 vam3Δ 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 vam3Δ 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. ​(Fig.10).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).

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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.

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.