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Mol Biol Cell. Nov 2004; 15(11): 5075–5091.
PMCID: PMC524777

Role of Vma21p in Assembly and Transport of the Yeast Vacuolar ATPase

Juan S. Bonifacino, Monitoring Editor

Abstract

The Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multisubunit complex composed of a peripheral membrane sector (V1) responsible for ATP hydrolysis and an integral membrane sector (V0) required for proton translocation. Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p. We found that in vma21Δ cells, the major proteolipid subunit of V0 failed to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly. Immunoprecipitation of Vma21p from wild-type membranes resulted in coimmunoprecipitation of all five V0 subunits. Analysis of vmaΔ strains showed that binding of V0 subunits to Vma21p was mediated by the proteolipid subunit Vma11p. Although Vma21p/proteolipid interactions were independent of Vph1p, Vma21p/Vph1p association was dependent on all other V0 subunits, indicating that assembly of V0 occurs in a defined sequence, with Vph1p recruitment into a Vma21p/proteolipid/Vma6p complex representing the final step. An in vitro assay for ER export was used to demonstrate preferential packaging of the fully assembled Vma21p/proteolipid/Vma6p/Vph1p complex into COPII-coated transport vesicles. Pulse-chase experiments showed that the interaction between Vma21p and V0 was transient and that Vma21p/V0 dissociation was concomitant with V0/V1 assembly. Blocking ER export in vivo stabilized the interaction between Vma21p and V0 and abrogated assembly of V0/V1. Although a Vma21p mutant lacking an ER-retrieval signal remained associated with V0 in the vacuole, this interaction did not affect the assembly of vacuolar V0/V1 complexes. We conclude that Vma21p is not involved in regulating the interaction between V0 and V1 sectors, but that it has a crucial role in coordinating the assembly of V0 subunits and in escorting the assembled V0 complex into ER-derived transport vesicles.

INTRODUCTION

Acidification of vacuoles/lysosomes, endosomes, and clathrin-coated vesicles is largely accomplished by the action of vacuolar-type proton-translocating ATPases (V-ATPases). Defects in V-ATPase function result in disruption of many cellular processes, including receptor-mediated endocytosis, targeting of lysosomal enzymes, zymogen activation, and uptake of small molecules through secondary transporters (Inoue et al., 2003 blue right-pointing triangle). The Saccharomyces cerevisiae V-ATPase is the most well-characterized member of this ubiquitous family of electrogenic pumps and is an excellent model for studying the biosynthesis of a multisubunit enzyme complex. The basic structure of V-ATPases is similar to mitochondrial F0F1-ATPases, consisting of an integral membrane V0 sector and a peripherally associated V1 sector. The V0 sector is composed of multiple transmembrane proteins that form a pathway for proton translocation, whereas the cytosolic V1 sector is assembled from soluble components that perform the catalytic functions of the enzyme. Genetic and biochemical analyses in yeast have identified 13 VMA (vacuolar membrane ATPase) genes that encode structural subunits of the V0/V1 complex.

Studies of vma mutants have revealed that V-ATPase assembly is in some ways a coordinated process (requiring the presence of other subunits) and is in other respects an autonomous process (proceeding in the absence of other V0 or V1 components). The cytosolic V1 sector is composed of at least eight subunits (69, 60, 54, 42, 32, 27, 14, and 13 kDa). With the exception of cells lacking Vma4p or Vma10p, the loss of a V1 subunit does not affect the stability of the remaining subunits, nor does it prevent their assembly into aV1 substructure (Tomashek et al., 1997 blue right-pointing triangle). However, with one exception, loss of Vma13p, these partially assembled V1 complexes fail to be recruited to the vacuolar membrane (Ho et al., 1993b blue right-pointing triangle). In cells lacking any one of the five gene products (100, 36, 23, 17, and 16 kDa) comprising the V0 sector, the V1 sector also fails to be recruited to the membrane (Bauerle et al., 1993 blue right-pointing triangle; Hirata et al., 1997 blue right-pointing triangle; Graham et al., 2003 blue right-pointing triangle).

V0 subunits are assembled and delivered to the vacuole independently of V1; however, the assembly and stability of V0 subunits is severely compromised in the absence of any of the other V0 components. Notably, the stability of the 100-kDa subunit, Vph1p, is drastically reduced in cells lacking other V0 subunits (Graham et al., 1998 blue right-pointing triangle). The half-time for Vph1p turnover is greater than 400 min in wild-type cells, compared with ~30 min in V0 mutants. Several lines of evidence indicate that Vph1p molecules that fail to assemble into V0 are degraded directly out of the endoplasmic reticulum (ER) via a proteasomal pathway (Hill and Stevens, 1995 blue right-pointing triangle; Hill and Cooper, 2000 blue right-pointing triangle; Wilhovsky et al., 2000 blue right-pointing triangle). The 36-kDa subunit (Vma6p) is a peripheral membrane protein that remains stable in V0 mutants but no longer associates with membranes. The highly hydrophobic 16-, 17-, and 23-kDa subunits (Vma11p, Vma3p, and Vma16p, respectively), termed proteolipids, have been difficult to characterize biochemically (Hirata et al., 1997 blue right-pointing triangle).

Genetic screens for vma mutants have also led to the isolation of three genes, VMA12, VMA21, and VMA22, that are required for V-ATPase biogenesis, but whose products are not structural components of V0V1 (Bachhawat et al., 1993 blue right-pointing triangle; Ho et al., 1993a blue right-pointing triangle; Hill and Stevens, 1994 blue right-pointing triangle). The biochemical phenotypes of vma12Δ, vma22Δ, and vma21Δ cells resemble those of strains lacking a structural subunit of V0: V0 fails to assemble properly, V0 subunits are not transported to the vacuole, and Vph1p is rapidly degraded via a proteasomal pathway (Hill and Stevens, 1995 blue right-pointing triangle; Hill and Cooper, 2000 blue right-pointing triangle; Wilhovsky et al., 2000 blue right-pointing triangle). Interestingly, immunofluorescence and cell fractionation studies showed that all three proteins localize to the ER, suggesting that they have a dedicated role in V0 biogenesis (Hill and Stevens, 1994 blue right-pointing triangle, 1995 blue right-pointing triangle; Jackson and Stevens, 1997 blue right-pointing triangle; Graham et al., 2003 blue right-pointing triangle).

Vma12p is a 25-kDa integral membrane protein predicted to contain two transmembrane segments (Jackson and Stevens, 1997 blue right-pointing triangle). Vma22p is a 21-kDa peripheral membrane protein whose membrane association is dependent on Vma12p (Hill and Stevens, 1995 blue right-pointing triangle). Indeed, Vma12p and Vma22p interact to form an assembly complex that binds newly synthesized Vph1p molecules in the ER (Graham et al., 1998 blue right-pointing triangle). Neither Vma21p nor other V0 subunits were found associated with the Vma12/22p assembly complex. Furthermore, the interaction between Vma12/22p and Vph1p persisted in vma21Δ cells and in cells lacking any one of the proteolipid subunits of V0 (Graham et al., 1998 blue right-pointing triangle). Together these findings suggest that Vma12/22p is dedicated to Vph1p maturation/assembly and that Vma21p might act at a subsequent stage in V0 assembly or in a parallel pathway.

VMA21 encodes an 8.5-kDa integral membrane protein that is predicted to span the membrane twice with both the amino- and carboxy-termini facing the cytosol. Intriguingly, the carboxy-terminus of Vma21p contains a -KKXX ER-retrieval sequence. Mutation of the -KKXX motif in Vma21p to -QQXX led to the mislocalization of Vma21(QQ)p to the vacuolar membrane (Hill and Stevens, 1994 blue right-pointing triangle). This observation suggests that Vma21p cycles between the ER and Golgi and that it may have a role in the transport of V0 subcomplex out of the ER. Interestingly, despite Vma21(QQ)p being mislocalized to the vacuole, Vma21(QQ)p is able to function in the assembly of the V-ATPase. Vacuoles prepared from vma21Δ cells expressing Vma21(QQ)p retain 30% of wild-type V-ATPase activity (Hill and Stevens, 1994 blue right-pointing triangle). These data indicate that Vma21p is required for V0 assembly and suggest that ER retrieval is important for Vma21p to act in multiple rounds of assembly.

In this article we use biochemical tools to further characterize the role of ER-localized accessory factors in V0 biogenesis. We found that Vma12p/Vma22p were not required for interaction of V0 subunits, but were essential for the productive assembly of these subunits into a functional V0 structure. In contrast, Vma21p was found to be required for the association of V0 subunits and was enriched as a complex with V0 during COPII vesicle formation.

MATERIALS AND METHODS

Yeast Strains, Growth Conditions, and Reagents

Yeast strains and plasmids used in this study are listed in Table 1. Yeast were transformed using a standard lithium acetate procedure (Schiestl and Gietz, 1989 blue right-pointing triangle). Yeast were grown in minimal (SD) or rich (YPD) medium (Sherman, 1991 blue right-pointing triangle) at 30°C or at 24°C for temperature sensitive strains. Vma- strains were grown in SD medium or in YPD that was buffered at pH 5 with 50 mM succinate/phosphate (Kane et al., 1992 blue right-pointing triangle).

Table 1.
Yeast strains and plasmids used in this study

Rabbit polyclonal antibodies raised against a 12× HA epitope (Bowers et al., 2000 blue right-pointing triangle) and against a maltose-binding protein (MBP) fusion to the N-terminal cytosolic domain (a.a. 1-369) of Vph1p (Hill and Stevens, 1994 blue right-pointing triangle) were generated as described. Purified mouse monoclonal antibodies against HA (HA.11) were obtained from BAbCo (Berkeley, CA). Mouse monoclonal antibodies against Vph1p (10D7), Vma1p (8B1), and Vma2p (13D11) were purchased from Molecular Probes (Eugene, OR). Rabbit polyclonal antibodies against CPY (Stevens et al., 1982 blue right-pointing triangle) and against Chs3p (Chuang and Schekman, 1996 blue right-pointing triangle) were also used in these experiments.

Metabolic Labeling and Pulse-chase Analysis

Yeast cells were grown to midlog phase, harvested, washed twice with SD media, and then grown in the absence of methionine for 15 min. [35S]methionine/[35S]cysteine labeling mix (35S-Promix, Amersham Biosciences, Piscataway, NJ) was added (6 μCi for each OD600 of cells harvested) and growth was continued for 3-60 min. Labeling was terminated by the addition of chase mix containing excess unlabeled amino acids (Chuang and Schekman, 1996 blue right-pointing triangle). Samples removed at various times during the chase period were arrested by the addition of sodium azide to 20 mM and incubation on ice. When all time points had been collected, cells were harvested by centrifugation and converted to spheroplasts with lyticase (Wuestehube and Schekman, 1992 blue right-pointing triangle).

Pulse-chase experiments designed to preserve the interaction between V0 and V1 were conducted using spheroplasts, as described in Kane (1995 blue right-pointing triangle).

Immunoprecipitations

For denaturing immunoprecipitations, 100 μl 1% SDS was added to sedimented spheroplasts. Samples were heated at 55°C for 5-10 min and 1 ml of dilution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM NaN3) containing protease inhibitors (1 mM PMSF, 1 μg/μl leupeptin, 1 μg/μl pepstatin, 2 μg/μl chymostatin, 5 μg/μl aprotinin) was added. Samples were placed on ice for ~10 min, and insoluble material was removed by centrifugation. Antibodies precoupled to either protein A- (Amersham Biosciences) or protein G-Sepharose (Sigma-Aldrich, St. Louis, MO) were added and mixed with membrane extracts for either 2 h at room temperature or overnight at 4°C. Immune complexes were washed three times with dilution buffer containing 0.1% SDS, once with Tris/NaCl (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM NaN3), and eluted with 2× sample buffer (2× SB; Ausubel et al., 1987 blue right-pointing triangle) with heating at 55°C for 10 min.

For nondenaturing immunoprecipitations, 1 ml of 1% C12E9 (Sigma-Aldrich) in PBS (containing protease inhibitors as described for dilution buffer) was used to solubilize sedimented spheroplasts. Protein A-Sepharose (50 μl) was added, and samples were mixed for 30 min at 4°C. Insoluble material was removed by centrifugation (12,000 × g, 15 min, 4°C) and immobilized antibody was incubated with the soluble material at 4°C for >4 h to overnight. Unbound material was removed by washing four times with 1% C12E9 in PBS and once with Tris/NaCl. Immune complexes were eluted with 2× SB and heating and analyzed by SDS-PAGE and phosphorimaging.

Nondenaturing immunoprecipitation of Vma1p was carried out as described in Kane (1995 blue right-pointing triangle) using monoclonal antibodies against Vma1p. Nondenaturing immunoprecipitation of Vma21p(QQ)-HA from solubilized vacuolar membranes was carried out as previously described by Powell et al. (2000 blue right-pointing triangle) using rabbit anti-HA sera.

In Vitro Assay for ER Export via COPII-coated Vesicles

Semi-intact yeast cells (SICs) and yeast microsomal membranes used in COPII packaging assays were prepared as described (Shimoni and Schekman, 2002 blue right-pointing triangle). COPII-coated vesicle packaging assays were performed as described in Kuehn et al. (1996 blue right-pointing triangle). Antibody inhibition experiments were performed as follows: SICs were sedimented, resuspended in 1 ml high acetate B88 (1 M KOAc, 20 mM HEPES-KOH, pH 6.8, 250 mM sorbitol, 5 mM Mg(OAc)2) and incubated on ice for 5 min. They were then washed twice with B88 (150 mM KOAc, 20 mM HEPES-KOH, pH 6.8, 250 mM sorbitol, 5 mM Mg(OAc)2) and brought to 50 OD/ml. Bovine serum albumin (BSA; Two hundred microliters of a 10% solution in B88) was added to the membranes before the addition of 10 μg of monoclonal antibodies against HA. Mock incubations were conducted in parallel and differed only in that anti-HA antibodies were omitted. Samples were mixed at 4°C for 90 min, and then unbound antibodies were removed by washing twice with 0.1% BSA in B88 and twice with B88. Membranes were then used in COPII-coated vesicle packaging assays.

Glycerol Gradient Fractionation of Vacuolar Membranes

Vacuolar membranes were prepared from vma21Δ cells expressing either Vma21p-HA (pKH28) or Vma21p(QQ)-HA (pKH31) as previously described (Conibear and Stevens, 2002 blue right-pointing triangle). Vacuolar membranes were resuspended in TE + 10% glycerol to a protein concentration of 5 mg/ml and solubilized with 1% C12E9 (final concentration). Solubilized proteins were fractionated on a 12.5 ml 20-50% glycerol gradient poured in tubes for a SW41 rotor after centrifugation at 175,000 × g for 20 h (Kane et al., 1989 blue right-pointing triangle). Fractions of ~0.75 ml were removed from the top of the gradient, analyzed by SDS-PAGE, and probed with antibodies specific to Vph1p, Vma2p, and HA.

RESULTS

Vma21p Is Required for V0 Assembly

Previous work has shown that Vph1p is rapidly degraded in cells lacking any one of the ER-localized accessory factors (vma12Δ, vma21Δ, or vma22Δ) required for V0 biogenesis (Hill and Stevens, 1994 blue right-pointing triangle, 1995 blue right-pointing triangle; Jackson and Stevens, 1997 blue right-pointing triangle). We speculated that in the absence of accessory factors, V0 subunits would fail to associate with each other, resulting in free Vph1p molecules being diverted for degradation. To test this, we assessed the interaction between Vph1p and the major proteolipid subunit of V0, Vma3p, by coimmunoprecipitation.

A fully functional VMA3::HA allele was integrated at the VMA3 locus in wild-type and vmaΔ strains (Hirata et al., 1997 blue right-pointing triangle). Transformants that expressed Vma3p-HA were grown to midlog phase and metabolically labeled with [35S]methionine for 60 min. Half of the cell extract was solubilized under denaturing conditions and used in immunoprecipitations with anti-Vph1p antibodies (Figure 1, Vph, odd numbered lanes). The remaining half was solubilized under nondenaturing conditions using a detergent (C12E9) known to preserve interactions within the fully assembled V0 complex (Doherty and Kane, 1993 blue right-pointing triangle). Immunoprecipitations with anti-HA antibodies were performed to recover Vma3p-HA (Figure 1, HA, even numbered lanes), and the amount of coimmunoprecipitated 100-kDa subunit was compared with that found in anti-Vph1p immunoprecipitations.

Figure 1.
Vma21p is required for V0 assembly. Wild-type and vmaΔ strains transformed with an HA-tagged version of the major proteolipid V0 subunit (VMA3::HA) were metabolically labeled with [35S]methionine for 60 min and harvested. Control cells lacking ...

Two control experiments presented in Figure 1 confirmed the specificity of the immunoprecipitations. In cells lacking HA-tagged Vma3p (lane 2), Vph1p was not directly recovered by anti-HA antibodies, as demonstrated by the absence of a 100-kDa band in anti-HA immunoprecipitations. Second, no significant 100-kDa band was detected in immunoprecipitations from vph1Δ cell extracts (lanes 5 and 6). This demonstrates two important points: that our Vph1p antibody does not recognize the alternative 100-kDa subunit, Stv1p; and that Stv1p is not a significant component of the 100-kDa band recovered in complex with Vma3p-HA. This is consistent with the fact that Stv1p is expressed at much lower levels than Vph1p and represents only a small fraction of total cellular 100-kDa V0 subunit (Manolson et al., 1994 blue right-pointing triangle).

Analysis of wild-type VMA3::HA cells showed that a prominent 100-kDa band was coimmunoprecipitated with Vma3p-HA (Figure 1, lanes 3 and 4). Quantification of these results revealed that ~80% of radiolabeled Vph1p was coimmunoprecipitated with Vma3p-HA. This indicates that the majority of Vph1p synthesized during the 60-min labeling period assembled into a V0 structure in Vma+ cells. In contrast, immunoprecipitations from cells lacking a proteolipid subunit of V0 (vma11Δ) showed that only ~2.6% of radiolabeled Vph1p was recovered in a complex with Vma3p-HA (Figure 1, lanes 7 and 8). Similar results were obtained in vma6Δ cells (Figure 1, lanes 9 and 10), where ~1.1% of Vph1p coimmunoprecipitated with Vma3p-HA, and in vma16Δ cells (unpublished data). Thus, Vph1p fails to interact with Vma3p if any one of the other three structural subunits of V0 is absent. This finding supports the idea that Vph1p requires association with other V0 subunits for its stability.

We next asked whether ER-accessory factors are required to facilitate the association of V0 subunits. In VMA3::HA vma21Δ cells, Vph1p failed to coimmunoprecipitate with Vma3p-HA, indicating that Vma21p is required for V0 assembly (Figure 1, lanes 11 and 12). Unexpectedly, we found that in vma12Δ cells, Vph1p was efficiently coimmunoprecipitated with Vma3p-HA (Figure 1, lanes 13 and 14). Approximately 70% of radiolabeled Vph1p in vma12Δ cells was complexed with Vma3p-HA. Identical results were observed in vma22Δ cells (unpublished data). Previous work has shown that Vma12p/Vma22p interacts with Vph1p during its biogenesis and that this interaction is required for Vph1p stability (Graham et al., 1998 blue right-pointing triangle). However, our findings indicate that Vph1p still interacts with other V0 subunits in the absence of Vma12p/Vma22p, but that this interaction is not sufficient to guard Vph1p from degradation.

Vma21p Interacts with V0 Subunits

The results from Figure 1 indicated that Vma21p plays a critical role in bringing together Vph1p and the proteolipid subunits of the V0 complex. To better understand how Vma21p facilitates this process, we asked whether Vma21p itself interacted with V0 subunits. The Vma12p/Vma22p complex is known to bind directly to Vph1p during V0 biogenesis, so we speculated that Vma21p might associate with the proteolipid subunits of V0 in a parallel pathway (Graham et al., 1998 blue right-pointing triangle).

To address this question, we took a similar strategy to that outlined for the experiments in Figure 1. Cells expressing a functional, HA-tagged version of Vma21p were radiolabeled with [35S]methionine for 20 min, membranes were solubilized under nondenaturing conditions, and Vma21p-HA was recovered by immunoprecipitation with antibodies against HA. Immunoprecipitated proteins were separated by SDS-PAGE and analyzed with a phosphorimager. The control experiment shown in lane 1 of Figure 2A, demonstrated that only a few radiolabeled proteins were nonspecifically immunoprecipitated from cells that lacked an HA-tagged protein. In contrast, immune complexes isolated from wild-type cells expressing Vma21p-HA contained a number of prominent radiolabeled proteins (Figure 2A, lane 2). The ~14-kDa protein at the bottom of the gel was identified as Vma21p-HA by performing anti-HA immunoprecipitations under denaturing conditions (unpublished data). Seven coimmunoprecipitated proteins were clearly present in immune complexes isolated from cells expressing Vma21p-HA and absent from the control sample (bands present in Figure 2A, lane 2 and not in lane 1). The electrophoretic mobility of five of these proteins corresponds exactly to the previously reported mobility of the five structural subunits of V0 (Vph1p, 100-kDa; Vma6p, 36-kDa; Vma16p, 23-kDa; Vma3p, 17-kDa; Vma11p, 16-kDa; Hirata et al., 1997 blue right-pointing triangle). We confirmed the identity of these V0 subunits by secondary immunoprecipitation using antibodies against Vph1p (unpublished data) and by analysis of vmaΔ strains (Figure 2A, lanes 3-7). To identify conclusively the three proteolipid species, we repeated the experiment described in Figure 2A using strains that express myc-tagged versions of Vma3p, Vma11p, and Vma16p. Addition of the myc-tag resulted in a shift in electrophoretic mobility that allowed us to clearly resolve each proteolipid species in Vma21p-HA immune complexes (Figure 2B). In addition to the five V0 subunits, two other prominent radiolabeled species were detected in Vma21p-HA immune complexes (Figure 2A, asterisks). The identity of these proteins was not determined, but their electrophoretic mobility does not correspond to that of Vma12p or Vma22p (Hirata et al., 1993 blue right-pointing triangle; Hill and Stevens, 1995 blue right-pointing triangle).

Figure 2.
Vma21p interacts with V0 subunits. (A) Wild-type and vmaΔ strains expressing Vma21p-HA were metabolically labeled with [35S]methionine for 20 min. Control strains possessing an untagged VMA21 allele were treated similarly. Cells were lysed and ...

Having found that all five structural components of V0 associate with Vma21p-HA, we wondered whether Vma21p interacts directly or indirectly with individual subunits. Therefore, we examined which V0 subunits coimmunoprecipitated with Vma21p-HA in a series of vmaΔ strains (Figure 2A, lanes 3-7). In vph1Δ cells expressing Vma21p-HA, only the 100-kDa band corresponding to Vph1p was absent from the collection of proteins immunoprecipitated from wild-type cells (compare lanes 2 and 3 in Figure 2A). Deletion of any of the other four structural components of V0 (vma6Δ, vma16Δ, vma3Δ, and vma11Δ, Figure 2A, lanes 4-7) also resulted in loss of Vph1p from Vma21p-HA immune complexes. Although Vph1p is destabilized in these strain backgrounds, resulting in reduced levels of total radiolabeled Vph1p (Figure 1, lanes 7-14), this instability cannot account for the absence of a significant 100-kDa band in Figure 2A, lanes 4-7. Therefore, the interaction between Vma21p and Vph1p appears to be very indirect, requiring that all other V0 subunits are present. Together with our observation that Vph1p fails to interact with Vma3p-HA in vma11Δ, vma16Δ, and vma6Δ cells (Figure 1 and unpublished data), we conclude that Vph1p is only incorporated into a nascent V0 structure when all other V0 subunits are preassembled in a complex with Vma21p.

As mentioned above, in vma6Δ cells expressing Vma21p-HA, the 100-kDa band corresponding to Vph1p was not detected in anti-HA immune complexes. As expected, the band at ~36-kDa corresponding to Vma6p, was also absent (Figure 2A, lane 4). However, all three proteolipid subunits were recovered along with Vma21p-HA in vma6Δ cells.

Analysis of strains lacking individual proteolipid subunits is shown in lanes 5-7 of Figure 2A. In all three proteolipid mutants, the Vph1p and Vma6p failed to coimmunoprecipitate with Vma21p-HA. This indicates that, akin to Vph1p, recruitment of Vma6p into V0 requires a preassembled Vma21p/proteolipid complex. In vma16Δ cells, we also failed to detect the 23-kDa subunit (Vma16p), but still observed coimmunoprecipitation of the Vma3p (17-kDa), and perhaps also Vma11p (16-kDa; obscured by smearing of the 17-kDa band; Figure 2A, lane 5). Similarly, the Vma16p and Vma11p were recovered in a complex with Vma21p-HA in the absence of Vma3p (Figure 2A, lane 6). However, in vma11Δ cells, Vma21p-HA did not coimmunoprecipitate any of the other V0 subunits, indicating that Vma11p is likely to provide the sole binding surface that allows Vma21p-HA to interact with other V0 subunits (Figure 2A, lane 7).

The experiment in Figure 1 yielded the unexpected result that Vph1p can interact with the major proteolipid subunit of V0 (Vma3p) in vma12Δ cells. Consistent with this observation, we found that all five V0 subunits were present in Vma21p-HA immune complexes isolated from vma12Δ cells (Figure 2A, lane 8). Identical results were obtained in vma22Δ cells (unpublished data). These results demonstrate that all five V0 subunits associate in vma12Δ and vma22Δ mutants, forming a complex whose composition is identical to that observed in wild-type cells, but whose structure or organization is aberrant.

Vma21p/V0 Association Is Transient and Is Stabilized by Blocking Exit from the ER

Vma21p is localized to the ER at steady state, whereas Vph1p and other V0 subunits are most abundant in the vacuolar membrane. Therefore, the interaction between Vma21p and V0 should be transient, occurring only during V0 biogenesis. We explored the dynamics of the association between Vma21p and the V0 complex using a pulse-chase protocol.

Cells expressing Vma21p-HA were grown overnight at 24°C, and then 5 min before labeling, cultures were split and incubated at either 24 or 37°C. Pulse labeling was initiated by the addition of [35S]methionine, and 5 min later terminated by the addition of an excess of nonradioactive amino acids. Aliquots of cells collected at various times during the chase period were solubilized under nondenaturing conditions. Anti-HA immune complexes isolated from these cell extracts were resolved by SDS-PAGE and analyzed using a phosphorimager. Vph1p that coimmunoprecipitated with Vma21p-HA is shown in Figure 3A. We inferred the kinetics of Vma21p/V0 interactions from coimmunoprecipitation of Vph1p with Vma21p. This simplification is justified from the experiments in Figure 2A, which demonstrated Vph1p only interacts with Vma21p when all other V0 subunits are bound to Vma21p. Therefore, coimmunoprecipitation of Vph1p represents coimmunoprecipitation of the V0 sector.

Figure 3.
The interaction between Vma21p and V0 is transient, but can be stabilized by blocking ER export. (A) Wild-type (RSY250/pKH28) and sec12-4 (RSY263/pKH28) strains expressing Vma21p-HA were grown at 24°C to midlog growth phase and then shifted to ...

The top panel of Figure 3A shows that in wild-type cells incubated at either 24 or 37°C, the amount of Vph1p complexed with Vma21p-HA decreases over the course of the chase. The kinetics of Vma21p/Vph1p dissociation are rapid, with only half of radiolabeled Vph1p recovered in complex with Vma21p-HA after 5.4 min of chase (Figure 3B). As might be expected, the kinetics of Vma21p/V0 dissociation is faster at 37°C (t1/2 ≈ 3.0 min). The decrease in the amount Vph1p coimmunoprecipitated with Vma21p-HA was not a result of Vph1p degradation, because no significant change was observed in the total amount of radiolabeled Vph1p present in cells during the 16-min chase period (unpublished data).

We extended this analysis by exploring the dynamics of Vma21p/V0 association in a sec12-4 strain that displays a conditional defect in the budding of secretory vesicles from the ER (Kaiser and Schekman, 1990 blue right-pointing triangle). The bottom panel of Figure 3A shows that Vph1p dissociated from Vma21p-HA in sec12-4 cells held at 24°C, but remained associated with Vma21p-HA when sec12-4 cells were incubated at 37°C. The graph in Figure 3B is a quantification of the data presented in Figure 3A.

These experiments demonstrated that the Vma21p/V0 complex we identified was not an artifact that occurs after cell lysis and loss of compartmental integrity. Rather, as radiolabeled Vph1p molecules moved through the secretory pathway to the vacuole, the interaction between Vma21p and Vph1p was lost. Furthermore, blocking transport from the ER prolonged the interaction between Vma21p and V0, suggesting that Vma21p may dissociate from the assembled V0 complex only after exiting the ER. The notion that Vma21p normally leaves the ER is supported by the presence of a bona fide ER-retrieval motif in its C-terminal cytosolic domain (Hill and Stevens, 1994 blue right-pointing triangle). We next entertained the possibility that Vma21p promotes packaging of the assembled V0 complex into COPII-coated transport vesicles, which carry secretory cargo out of the ER.

The Vma21p/V0 Complex Is Packaged into COPII-coated Vesicles

We used a well-characterized in vitro assay that recapitulates ER export to determine if Vma21p is packaged into COPII-coated vesicles (Shimoni and Schekman, 2002 blue right-pointing triangle). Briefly, microsomal membranes were purified from cell extracts and were combined with purified COPII proteins to generate ER-derived transport vesicles. Total and vesicle fractions were collected and then analyzed by SDS-PAGE and Western blotting.

In Figure 4A, microsomal membranes from cells expressing Vma21p-HA or Vma12p-HA were used in our in vitro ER-export assay. Packaging of Sec61p, the major subunit of the translocon and an ER-resident protein, served as a negative control. Almost no Sec61p could be detected in the vesicle fraction generated from either source of microsomal membranes. In contrast, Sec22p, an R-SNARE that cycles between the ER and Golgi, was efficiently captured into COPII-coated vesicles. Vma21p-HA was easily detected in the COPII-coated vesicle fraction, but was packaged less efficiently than Sec22p. Unlike Vma21p-HA, Vma12p-HA was barely detectable in the vesicle fraction. This finding is consistent with a primary role for Vma12p in the biogenesis of Vph1p, a role that is restricted to the ER.

Figure 4.
Vma21p and Vph1p are packaged into COPII-coated vesicles. (A) Microsomal membranes prepared from cells expressing either Vma21p-HA or Vma12p-HA were incubated with GTP, and COPII proteins at 25°C for 30 min. Total (10%) and vesicle (60%) fractions ...

Having found that Vma21p could be packaged into COPII-coated vesicles in vitro, we wanted to know if the complex containing Vma21p and the fully assembled V0 sector (see Figure 2A) was preferentially taken-up into COPII-coated vesicles. Unfortunately, the microsomal membranes used in the ER-export assay described above are heavily contaminated with vacuolar membranes, which are the primary cellular location of V0 subunits. Therefore, we turned to an ER-export assay that measures only the packaging of newly synthesized proteins into COPII-coated vesicles (Kuehn et al., 1996 blue right-pointing triangle). Cells were grown to midlog phase and labeled with [35S]methionine for 3 min. Sodium azide was then added to arrest metabolic activity and, thus, prevent the ER export of membrane proteins that had incorporated radiolabeled amino acids during their synthesis. After converting cells to spheroplasts, we gently ruptured the plasma membrane to yield semi-intact cells (SICs), and these were used for in vitro ER-export reactions, similar to those described in Figure 4A. Radiolabeled proteins were recovered by immunoprecipitation, resolved by SDS-PAGE, and analyzed with a phosphorimager.

We first sought to determine if this assay could faithfully measure Vph1p packaging into COPII-coated vesicles. As shown in Figure 4B (top panel), radiolabeled Vph1p was released into the vesicle fraction (11.6% of total) when COPII proteins and GTP were present in the ER-export assay. GTP is required to activate the small GTP-binding protein, Sar1p, which is a component of the COPII budding machinery (Barlowe et al., 1993 blue right-pointing triangle). In control reactions lacking GTP, only 2.6% of radiolabeled Vph1p was taken-up into vesicles. To determine if the ER-export efficiency we observed for Vph1p was similar to that of other known secretory cargo, we analyzed the packaging of Gap1p-HA and Chs3p. We chose these proteins because, like Vph1p, both Gap1p and Chs3p have multiple membrane-spanning segments and depend on ER-localized accessory factors to orchestrate their biogenesis and ER export (Ljungdahl et al., 1992 blue right-pointing triangle; Trilla et al., 1999 blue right-pointing triangle). We found that Gap1p-HA was packaged into COPII-coated vesicles with an efficiency similar to that of Vph1p (Figure 4B). In reactions containing GTP, the percentage of total Gap1p-HA in the vesicle fraction was 11.0%. Chs3p was also released into the vesicle fraction in a GTP-dependent manner. The packaging efficiency of Chs3p was slightly lower (7.6%) than that observed for Vph1p and Gap1p. This may be due to the longer labeling time (10 min) required to obtain detectable amounts of Chs3p.

We then used this packaging assay to determine if the Vma21p/V0 complex is taken-up into COPII-coated vesicles. Total and vesicle fractions, taken from reactions containing COPII proteins and GTP, were solubilized under nondenaturing conditions to preserve the interactions between V0 constituents. Anti-HA antibodies were used to immunoprecipitate Vma21p-HA and associated molecules (as described in Figure 2A). The results from this experiment are shown in Figure 5A. Consistent with the results in Figure 4A, Vma21p-HA is released into the vesicle fraction. The spectrum of radiolabeled proteins that coimmunoprecipitated with Vma21p-HA was the same in both the total and vesicle fractions; however, the stoichiometry of the these interacting proteins was different. The amount of proteolipid subunits complexed with Vma21p-HA was similar in both fractions (most clearly seen for Vma16p), whereas the amount of Vph1p coimmunoprecipitated with Vma21p-HA was significantly greater in the vesicle fraction. This suggested that the complex of Vma21p-HA and the fully assembled V0 sector was preferentially incorporated into COPII-coated vesicles.

Figure 5.
The Vma21p/V0 complex is enriched in COPII-coated vesicles. (A) Metabolically labeled SICs prepared from vma21Δ/VMA21-HA cells were used in a COPII-coated vesicle packaging assay as described in Figure 4B. Total (10%) and vesicle (70%) fractions ...

To quantify the enrichment of the Vma21p/V0 complex in vesicles, the percentage of total radiolabeled Vph1p that could be coimmunoprecipitated with Vma21p-HA was determined in both the total and vesicle fractions. ER-export assays using radiolabeled SICs and purified COPII proteins were performed as in Figure 5A. Aliquots (10%) of both the total and vesicle fractions were set aside, and the amount of Vph1p present in these samples was determined by denaturing immunoprecipitation using antibodies against Vph1p. The remaining 90% of the total and vesicle fractions were subjected to nondenaturing immunoprecipitation with anti-HA antibodies. As shown in Figure 5B, ~16% of radiolabeled Vph1p was complexed with Vma21p-HA in the total fraction, whereas ~37% of radiolabeled Vph1p was complexed with Vma21p-HA in the vesicle fraction. Thus, there was a twofold enrichment of the fully assembled Vma21p/V0 complex in COPII-coated vesicles.

Vma21p Is Required for ER Export of Vph1p In Vitro

If Vma21p plays an active role in directing V0 subunits into COPII-coated vesicles, these proteins should be inefficiently packaged into vesicles in the absence of Vma21p. To examine this possibility using our in vitro assay, we prepared radiolabeled SICs from wild-type and vma21Δ strains. Radiolabeled membranes were also isolated from vma11Δ strains, in which Vph1p fails to associate with other V0 subunits (Figure 1), and from vma12Δ strains, in which V0 subunits still associate, but form an aberrant complex (Figures (Figures11 and and2B).2B). These membranes were used in reactions containing COPII proteins and + or -GTP, and the percentage of Vph1p packaged into COPII-coated vesicles was calculated by comparing total and vesicle fractions, as in Figure 4B. Packaging of Chs3p, whose ER export is independent of VMA genes, was measured to control for variations in the activity of membrane preparations from different strains.

As shown in Figure 6 (left panel), packaging of Chs3p into COPII-coated vesicles was GTP dependent and occurred with efficiency similar to that of wild-type, vma11Δ, vma21Δ, and vma12Δ membranes. ER export of Vph1p was also GTP dependent, but varied greatly depending on the source of membranes. Approximately 9.3% of radiolabeled Vph1p was captured into COPII-coated vesicles from wild-type membranes, whereas packaging of Vph1p from vma11Δ and vma21Δ membranes measured only ~1.5 and ~1.4%, respectively. Interestingly, packaging of Vph1p from vma12Δ membranes was quite efficient (~5.1%). In experiments described in Figure 2A, we observed that Vph1p associates with all five V0 subunits and with Vma21p in vma12Δ cells. Therefore, efficient ER export of Vphp1 may rely primarily on alliance with other V0 subunits. The poor budding of Vph1p we observed in vma21Δ cells could be largely due to a V0 assembly defect, rather than indicative of a direct role for Vma21p in Vph1p packaging.

Figure 6.
Packaging of Vph1p into COPII-coated vesicles in vitro is dependent on VMA21. Radiolabeled membranes were prepared from wild-type, vma11Δ, vma21Δ, and vma12Δ cells and used in packaging assays as described in Figure 4B. Reactions ...

Anti-HA Antibodies Diminish Vph1p Budding from Vma21p-HA Membranes

To test whether Vma21p is involved in guiding the assembled V0 structure into transport vesicles, we attempted to inhibit Vma21p function after V0 assembly was complete. Membranes were pretreated with antibodies that bind Vma21p and then used in ER-export assays. We anticipated that the antibodies might obscure transport signals present on Vma21p, thereby reducing capture of the assembled V0 sector in COPII-coated vesicles.

Radiolabeled membranes were prepared from cells possessing an HA-tagged allele of VMA21 or VMA12 and from control cells lacking an HA-tagged protein. Before the addition of COPII proteins and GTP, membranes were incubated with anti-HA antibodies or mock treated with BSA. Total and vesicle fractions were isolated from ER-export reactions, and the percentage of Vph1p in the vesicle fraction was calculated. We found that the antibodies had no effect on Vph1p packaging from wild-type or Vma12p-HA membranes (Figure 7A). In contrast, preincubation of Vma21p-HA membranes with anti-HA antibodies resulted in ~37% inhibition of Vph1p packaging into COPII-coated vesicles (Figure 7A). Although modest, this inhibition of Vph1p budding was reproducible and specific to membranes containing Vma21p-HA. Control reactions conducted in parallel also demonstrated that incubation of Vma21p-HA containing membranes with anti-HA antibodies had no effect on packaging of a soluble secretory protein, glycosylated proalpha-factor (gpα-factor; Figure 7B). These results are consistent with the proposal that Vma21p plays a role in guiding the assembled V0 structure into vesicles that transport cargo out of the ER.

Figure 7.
Pretreatment of membranes with antibodies that recognize Vma21p reduces packaging of Vph1p into COPII-coated vesicles. (A) 35S-methionine-labeled SIC membranes were prepared from wild-type (SF838-1Dα), vma12Δ/VMA12::HA, and vma21Δ/ ...

Dissociation of Vma21p-V0 Precedes Assembly of V0/V1

We were intrigued by the possibility that Vma21p could serve another purpose in V-ATPase biogenesis: preventing premature assembly of an enzymatically active V0/V1 structure early in the secretory pathway. It is well known that there is a gradient of acidity across the secretory pathway in mammalian cells, with the pH of the ER lumen being neutral (~pH 7.2), the Golgi lumen mildly acidic (pH 7.0-6.2), and secretory granules quite acidic (~pH 5; Kim et al., 1996 blue right-pointing triangle; Miesenbock et al., 1998 blue right-pointing triangle; Wu et al., 2000 blue right-pointing triangle). Acidification of these organelles depends on the action of the V-ATPase; however, the basis of the pH gradient is not well understood (Nishi and Forgac, 2002 blue right-pointing triangle). In yeast it is presumed that a similar gradient exists along the secretory pathway, but only the pH of the lumen of the vacuole has been measured, and the vacuole lumen was found to be pH ~5.9 (Preston et al., 1989 blue right-pointing triangle; Plant et al., 1999 blue right-pointing triangle). One mechanism to establish such a gradient is to limit or prohibit the activity of the V-ATPase in early cellular compartments. Because Vma21p associates with V0 early in the secretory pathway, we hypothesized that this interaction might prevent assembly of a proton translocating V0/V1 structure in the ER and proximal Golgi, thereby reducing the acidity of these organelles.

To explore this possibility, we first compared the kinetics of Vma21p/V0 dissociation with the kinetics of V0/V1 assembly. In the pulse-chase experiment shown in Figure 8 (lanes 1-3) samples from a single culture of VMA21::HA cells were split and analyzed by immunoprecipitation for carboxypeptidase Y (CPY; panel 1), Vph1p (panel 2), Vph1p coimmunoprecipitated with Vma21p-HA (panel 3), Vph1p coimmunoprecipitated with Vma1p (the 69-kDa V1 subunit; panel 4), and Vma2p (the 60-kDa subunit of V1) coimmunoprecipitated with Vma1p (panel 5). As expected, CPY was proteolytically cleaved to a mature form during the course of the chase, indicating that it was transported to the vacuole (Conibear and Stevens, 2002 blue right-pointing triangle). The total amount of radiolabeled Vph1p did not change substantially (Figure 8, panel 2), whereas the amount of Vph1p associated with Vma21p-HA decreased during the chase period (Figure 8, panel 3). The rate of V0/V1 assembly, as assessed by the association of Vph1p (a V0 subunit) with Vma1p (a V1 subunit), increased during the chase. Quantification of these data reveals that the rate of Vma21p/V0 dissociation mirrored the rate of V0/V1 assembly, suggesting that Vma21p may free V0, allowing V0 to interact with the V1 sector (Figure 8B).

Figure 8.
Blocking secretion stabilizes the Vma21p/V0 complex and prevents V0/V1 assembly. (A) Wild-type (RSY250/pKH28) and sec18-1 (RSY271/pKH28) strains expressing Vma21p-HA were grown at 24°C and then shifted to the indicated temperature for 5 min before ...

The data presented in Figure 3 showed that blocking exit from the ER stabilized the interaction between Vma21p and V0. Once again we used a sec mutant, in which transport was conditionally arrested, to determine if stabilization of the Vma21p/V0 complex affected V0/V1 assembly. The right-hand panel of Figure 8A shows a pulse-chase experiment using sec18-1/VMA21::HA cells incubated at either 24 or 37°C. In sec18-1 cells grown at a restrictive temperature (37°C), transport vesicles fail to fuse with acceptor compartments, causing a rapid back-up in the secretory pathway that abrogates ER export (Novick et al., 1981 blue right-pointing triangle). As shown in Figure 8A (panel 1, lanes 7-12), transport and maturation of CPY occurred normally in sec18-1/VMA21::HA cells incubated at 24°C, but was fully eliminated at 37°C. The stability of Vph1p was not significantly affected by either the sec18 mutation or elevated temperature (Figure 8A, panel 2, lanes 7-12). In keeping with the results of Figure 3, dissociation of Vph1p from Vma21p-HA occurred rapidly in sec18 cells incubated at 24°C, but was completely blocked at 37°C (Figure 8A, panel 3, lanes 7-12). The amount of Vph1p coimmunoprecipitated with Vma1p increased steadily over the chase period when cells were incubated at 24°C. However, at 37°C, a consistently small amount of Vph1p coimmunoprecipitated with Vma1p, indicating that V0/V1 assembly was arrested by incubation of sec18 cells at 37°C (Figures (Figures8A,8A, panel 4, lanes 7-12, and and8C).8C). Association of V1 subunits with each other was not affected by the transport block, as demonstrated by uniform coimmunoprecipitation of Vma2p (the 60-kDa subunit of V1) with Vma1p in both wild-type and sec18 cells (Figure 8A, panel 5).

Vma21(QQ)p Associates with V0 in Vacuole, but Does Not Prevent V0/V1 Assembly

The data presented in Figure 8 argue that assembly of a V0/V1 complex may occur only after the release of the V0 sector from Vma21p. To further examine this possibility, we made use of a mutant form of Vma21p that is not retrieved to the ER, Vma21(QQ)p (Hill and Stevens, 1994 blue right-pointing triangle). We asked whether Vma21(QQ)p remains associated with V0 en route to the vacuole, and if so, whether Vma21(QQ)p/V0 interaction precludes formation of V0/V1 complexes.

First, we determined whether Vma21(QQ)p remains associated with the V0 subcomplex after leaving the ER. To do this, we performed a pulse-chase experiment comparing the half-life of the interaction between the V0 100-kDa subunit and wild-type or mutant Vma21p. vma21Δ cells expressing either Vma21p-HA or Vma21(QQ)p-HA were incubated for a brief time in the presence of radiolabel, followed by a 60-min incubation in the presence of excess unlabeled amino acids. Vma21p-HA and Vma21(QQ)p-HA were immunoprecipitated under nondenaturing conditions using an anti-HA antibody and analyzed using SDS-PAGE for coimmunoprecipitation of the 100-kDa V0 subunit. As shown in Figure 9A, very little 100-kDa subunit was coimmunoprecipitated with Vma21p after the 60-min chase, indicating that wild-type Vma21p interacts transiently with the 100-kDa V0 subunit. In contrast, the interaction between mutant Vma21p(QQ) and V0 was long-lived and did not appear to decrease after the 60-min chase period, suggesting that most of the newly synthesized 100-kDa subunit remains bound to Vma21p(QQ) even after V0 assembly is complete. However, this type of experiment does not tell us where in the cell the Vma21(QQ)p/V0 interaction occurs and whether these complexes are present on the vacuole or only en route to the vacuole?

Figure 9.
Vma21p(QQ) associates with V0 in vacuole, but does not prevent V0V1 assembly. (A) Cells expressing Vma21p-HA (vma21Δ/pKH28) or Vma21(QQ)p-HA (vma21Δ/pKH31) were metabolically labeled for 10 min and then chased for 60 min. Samples collected ...

We wanted to determine how much of the Vma21(QQ)p present on the vacuole interacts with the V0 subcomplex and if this continued interaction prevents V0/V1 assembly. Vacuolar membranes from cells expressing wild-type or mutant Vma21p were isolated, solubilized, and fractioned to determine if vacuolar Vma21p(QQ) cofractionates with V0 subunits and/or with V0/V1 complexes. Consistent with previously published immunolocalization of Vma21(QQ)p, considerably more of the mutant Vma21p was present on the vacuole compared with wild-type Vma21p (Figure 9B; compare the 3rd and 6th panels). As anticipated, a very low level of wild-type Vma21p was detected in fractions 4 and 5, and very likely represents contamination of the enriched vacuolar membranes with ER membranes (Jackson and Stevens, 1997 blue right-pointing triangle). Both V0 and V1 subunits were detected in fractions 7-11, indicating that these fractions contain fully assembled V-ATPase complexes (V0/V1). The low level of wild-type Vma21p did not cofractionate with the V0/V1 complex fractions, consistent with its presence in the vacuole-enriched sample due to contaminating ER membranes. A large proportion of the Vma21(QQ)p present in the vacuole (fractions 2-4) did not cofractionate with the V0/V1 containing fractions (7-11). This free Vma21(QQ)p may represent protein that has already dissociated from V0 subunits or Vma21(QQ)p that escaped the ER without being associated with V0 cargo. Importantly, at least some of the Vma21(QQ)p localized on the vacuole cofractionated with V0/V1 complexes (fractions 7-11), suggesting a direct interaction between Vma21(QQ)p and V0/V1 complexes on the vacuole.

To confirm whether Vma21(QQ)p is physically associated with the fully assembled V-ATPase complex on the vacuole membrane, we immunoprecipitated Vma21(QQ)p-HA from solubilized vacuole membranes and determined whether V0 and V1 subunits coprecipitated. Vacuole membranes were solubilized in 1% C12E9 and Vma21(QQ)p-HA precipitated with anti-HA polyclonal antibodies. The immunoprecipitated proteins were subjected to SDS-PAGE, and blots were probed with anti-HA monoclonal, anti-Vph1p (V0 subunit), and anti-Vma2p (V1 subunit) antibodies (Figure 9C). The presence of both Vph1p and Vma2p in the anti-HA immunoprecipitate of cells expressing Vma21(QQ)p-HA but not Vma21p indicates that Vma21(QQ)p is directly associated with the fully assembled V-ATPase complex on the vacuole membrane. These data indicate that the prolonged association of Vma21(QQ)p with V0 does not prevent assembly of the V1 subunits with the V0 subcomplex to form a fully assembled V-ATPase complex.

DISCUSSION

The ER is the site of membrane protein biosynthesis and, therefore, possesses a number of resident proteins that assist in the folding, assembly, and transport of newly translated membrane proteins. Some ER-resident chaperones in yeast act on a broad spectrum of targets (e.g., Kar2p, PDI), whereas others facilitate the biosynthesis of only a few substrates (Ellgaard et al., 1999 blue right-pointing triangle). Although a number of substrate-specific chaperones have been identified, the precise function of these proteins is largely unclear (Herrmann et al., 1999 blue right-pointing triangle). We sought to understand how three ER-localized accessory factors, (Vma12p, Vma21p, and Vma22p) facilitate biogenesis of the yeast V-ATPase.

Role of ER-localized Accessory Proteins in Assembly of the V0 Sector

Previous work has shown that the major 100-kDa subunit of V0 (Vph1p) is rapidly degraded in cells lacking either a V0 subunit or one of the V-ATPase accessory factors. It has been proposed that Vph1p instability in vma mutants is a result of a V0 assembly defect (Graham et al., 2003 blue right-pointing triangle). We tested this possibility by determining whether Vph1p could be coimmunoprecipitated with an HA-tagged version of the proteolipid subunit, Vma3p, in wild-type and vma mutant strains. As expected, the majority of Vph1p was complexed with Vma3p-HA in wild-type cells. In cells lacking the proteolipid subunit Vma11p or the peripheral subunit Vma6p, Vph1p failed to interact with Vma3p-HA. A striking defect in the association of Vph1p and Vma3p-HA was also observed in vma21Δ cells. Thus, one clear role for Vma21p in V-ATPase biogenesis is simply to bring the subunits of V0 together.

In vma12Δ and vma22Δ cells, Vph1p stability is severely comprised, yet Vph1p still associated with Vma3p-HA and other V0 subunits. This surprising result shows that a careful distinction must be made between association/interaction of V0 subunits and productive assembly of a V0 sector. Association of V0 subunits alone is not sufficient to create a functional V0 sector, nor is it sufficient to prevent Vph1p degradation.

It is possible that Vph1p is misfolded in vma12Δ and vmaΔ22 cells such that it still interacts with other V0 subunits, but is nonetheless targeted for rapid degradation. Previous analysis of the folded state of Vph1p by partial proteolysis yielded identical patterns when microsomes from wild-type and vmaΔ12 cells were exposed to protease (Jackson and Stevens, 1997 blue right-pointing triangle), suggesting no dramatic changes in the overall structure of the large N-terminal cytosolic domain of Vph1p. If Vph1p is misfolded in vma12Δ cells (and presumably vma22Δ cells), the effect must be subtle, because it still associates with other V0 subunits in the ER yet the complex formed is not competent for assembly with V1 or for transport to the vacuole.

More recent results provide evidence that in vma12Δ and vma22Δ cells Vph1p is degraded by a proteasomal pathway devoted to the removal of misfolded cytosolic proteins. Hill and Cooper found that UBC6 and UBC7, genes encoding ubiquitin conjugating enzymes required for degradation of misfolded integral membrane proteins, were not needed for degradation of Vph1p in vma22Δ cells (Hill and Cooper, 2000 blue right-pointing triangle). Instead, UBC1 and UBC4, two genes required for degradation of short-lived cytosolic proteins, influenced the rate of Vph1p degradation in vma22Δ cells. Furthermore, they found that cytosolic (Ssa1p), rather than lumenal (Kar2p), chaperones were involved in Vph1p delivery to the proteasome in vma22Δ cells. Curiously, other studies have shown that rapid degradation of Vph1p in vma21Δ cells depends on UBC6 and UBC7 as well as components of the ERAD machinery (Hrd1p; Wilhovsky et al., 2000 blue right-pointing triangle). At the time these data appeared contradictory, and the authors did not comment on the inconsistency between vma22Δ and vma21Δ strains. Given the results of Figures Figures11 and and2,2, it seems likely that Vph1p is in different assembly states in vma22Δ and vma21Δ cells and that this difference may influence the pathway that Vph1p takes to the proteasome. Therefore, we reason that Vma12p/Vma22p may be required for proper folding of the large cytosolic amino-terminal domain of Vph1p.

The absence of Vma21p from the Vma12p/Vma22p/Vph1p assembly complex suggests that Vma21p is involved in a parallel pathway (interacting with the proteolipid subunits) or in a downstream pathway (interacting with both Vph1p and other V0 subunits; Graham et al., 1998 blue right-pointing triangle). To explore this question, we asked what V0 subunits interacted with Vma21p-HA. We found that all five V0 subunits were specifically coimmunoprecipitated with Vma21p-HA. The interaction between Vma21p-HA and V0 was transient, reflecting the different cellular localizations of Vma21p and V0. Furthermore, blocking export of newly synthesized proteins out of the ER stabilized the interaction between Vma21p-HA and V0.

We used vmaΔ strains to determine which V0 subunits interact directly with Vma21p-HA and which V0 subunits associate with Vma21p-HA through other V0 subunits. We found that in vma3Δ and vma16Δ strains, the remaining two proteolipid subunits retained the ability to interact with Vma21p-HA, but Vma6p and Vph1p did not. In vma11Δ strains, no V0 subunits coimmunoprecipitated with Vma21p-HA. This suggests that Vma11p serves as the link that connects Vma21p-HA to V0. Vma21p is only 8.5-kDa, consisting of two transmembrane domains, a 12-residue lumenal loop, and N- and C-terminal cytosolic domains of 12 and 16 residues, respectively. It is somewhat remarkable that such a small molecule can coordinate the assembly of the ~240-kDa V0 sector, and it would be even more remarkable if it made contacts with each V0 subunit. Instead, Vma21p seems to interact with V0 via a single subunit, Vma11p.

The analysis of vmaΔ strains also suggests that Vma6p and Vph1p can only interact with Vma21p when all the proteolipid subunits are present. Although Vma6p coimmunoprecipitated with Vma21p-HA in vph1Δ cells, Vph1p was absent from Vma21p-HA/proteolipid complexes in vma6Δ cells. Thus, Vph1p can only associate with Vma21p-HA when all other V0 subunits are present. Together these observations suggest that Vma21p serves as a nucleation point around which V0 assembly takes place and that Vph1p is the last subunit added to form a completed V0 sector (Figure 10).

Figure 10.
A model for assembly and transport of the yeast V-ATPase. The Vma12p/Vma22p complex interacts transiently with newly synthesized Vph1p, perhaps aiding in folding the large cytosolic amino-terminal domain of this 100-kDa subunit. In a parallel pathway, ...

Transport of the Assembled V0 Sector out of the ER

The carboxy-terminal region of Vma21p contains a -KKXX sequence that is required for localization of Vma21p to the ER. Mutation of this sequence to -QQXX results in a protein, Vma21p-QQ, that accumulates in the vacuole (Hill and Stevens, 1994 blue right-pointing triangle). Many studies have shown that carboxy-terminal -KKXX sequences are directly recognized by components of the COPI coat and that binding leads to inclusion of -KKXX containing proteins into COPI-coated transport vesicles that travel from the Golgi toward the ER (Wieland and Harter, 1999 blue right-pointing triangle). The presence of a bona fide ER-retrieval signal in Vma21p suggests that Vma21p escorts the assembled V0 sector to the Golgi.

We used a well-established in vitro assay to examine the role of Vma21p in ER export of V0. Preliminary experiments showed that Vma21p-HA, but not Vma12p-HA, was packaged into ER-derived transport vesicles. This finding is consistent with the proposed role for Vma12p in facilitating folding of the amino-terminal domain of Vph1p, which most likely occurs in the ER during translation. Subsequent experiments examined packaging of Vma21p-containing protein complexes into COPII-coated vesicles. We found that the complex of Vma21p-HA and the fully assembled V0 sector was enriched approximately twofold in ER-derived vesicles. This suggests that ER export of V0 subunits is suspended until V0 assembly is complete and that Vma21p accompanies the V0 sector to the Golgi. This is the first demonstration of enrichment into ER-derived vesicles of a complex containing a transmembrane cargo complex (V0) and an accessory protein (Vma21p). Other studies have presented evidence for a physical interaction between a cargo protein and an accessory protein, but none of these studies showed preferential incorporation of a cargo-accessory complex into COPII-coated vesicles (Gas1p/p24, Muniz et al., 2000 blue right-pointing triangle; SREBP/SCAP, Nohturfft et al., 2000 blue right-pointing triangle; and gpα-factor, Erv29p, Belden and Barlowe, 2001 blue right-pointing triangle), Axl2p/Erv14p, Powers and Barlowe, 2002 blue right-pointing triangle). Importantly, several of these studies did demonstrate that specific amino acid sequences in the accessory protein were required for ER export of both cargo and escort. We have yet to identify such sequences in Vma21p.

To determine if Vma21p contains information required for ER export of the assembled V0 sector, we first examined packaging of Vph1p into COPII-coated vesicles in vma21Δ strains. Using our in vitro assay, we showed that ER export of Vph1p from vma21Δ membranes is about six times less efficient than from wild-type membranes. A similar defect in Vph1p packaging was observed when vma11Δ membranes were used. We showed in Figure 1 that both Vma21p and Vma11p are required for Vph1p to associate with other V0 subunits. Therefore, inefficient packaging of Vph1p in vma21Δ cells is most likely due to an assembly defect, rather than a direct role for Vma21p in Vph1p transport from the ER.

Interestingly, ER export of Vph1p from vma12Δ membranes was about three times more efficient than from vma21Δ or vma11Δ membranes. We found that Vph1p still associates with other V0 subunits and with Vma21p in vma12Δ cells (Figures (Figures11 and and2B).2B). This indicates that oligomerization of V0 subunits may be one factor that promotes V0 packaging. Individually, V0 subunits may display weak ER-export signals, that when brought together into a V0 sector, provide a sufficient impetus for inclusion into COPII-coated vesicles. Oligomerization has also been implicated in regulating transport of ERGIC-53 (Appenzeller et al., 1999 blue right-pointing triangle), Erv41p/Erv46p (Otte and Barlowe, 2002 blue right-pointing triangle), and Emp24p/Erv25p (Belden and Barlowe, 1996 blue right-pointing triangle).

To address the role of Vma21p in ER export of a properly assembled V0 sector, we tried to inhibit Vma21p function in wild-type membranes using antibodies. In these experiments, pretreatment of membranes with antibodies that recognize Vma21p reduced the ER export of Vph1p by ~37%. Although, this effect was modest, it was reproducible, specific to Vph1p (gpα-factor packaging was unaffected) and specific to membranes expressing an epitope-tagged Vma21p molecule recognized by the antibodies. We propose that the bound antibody obscured a transport signal required for efficient ER export of Vma21p/V0. It is unclear whether this transport signal is present on Vma21p or on an adjacent V0 subunit that might have also been obscured by bound antibody. To convincingly demonstrate a postassembly role for Vma21p in V0 transport, we are attempting to generate a Vma21p mutant, where assembly and transport functions are uncoupled.

Dissociation of Vma21p-V0 Complex and Assembly of V0/V1

We wondered whether Vma21p might also serve a regulatory function, preventing premature assembly of a proton-pumping V0/V1 complex early in the secretory pathway. In so doing, Vma21p could contribute to the maintenance of neutral pH in the ER and cis-Golgi compartments. Previous work has shown that coimmunoprecipitation of Vph1p (a V0 subunit) with Vma1p (a V1 subunit) is reduced in sec18-1 cells incubated at a restrictive temperature (Kane et al., 1999 blue right-pointing triangle). Surprisingly, the authors did not interpret this as evidence that V0/V1 complexes fail to assemble when transport from the ER is blocked. We extended their analysis by comparing the kinetics of Vma21p-HA/V0 dissociation to the kinetics of V0/V1 assembly in wild-type and sec18-1 cells. Pulse-chase experiments showed that the rate of Vma21p-HA/V0 dissociation mirrored the rate of V0/V1 assembly. Furthermore, blocking vesicle traffic arrested both dissociation of Vma21p/V0 and assembly of V0/V1. Our results indicate that V0/V1 cannot assemble on the ER membrane and that the interaction of Vma21p with V0 in early cellular compartments may block V0/V1 assembly.

To explore this possibility, we analyzed Vma21p/V0 dissociation and V0/V1 assembly in strains expressing a retrieval-defective Vma21p mutant, Vma21(QQ)p. Vma21(QQ)p was found to remain associated with V0 because both proteins transited the secretory pathway to the vacuole and in the vacuole as well. Surprisingly, the prolonged association of Vma21(QQ)p with the V0 subcomplex did not appear to have a significant effect on V0/V1 assembly. Gradient fractionation of vacuoles from strains expressing Vma21(QQ)p suggested that Vma21(QQ)p interacts with both free V0 sectors as well as assembled V0/V1 structures, and coprecipitation of V1 and V0 subunits with Vma21(QQ)p from vacuole membranes confirmed the direct association. Therefore, it seems unlikely that Vma21p prevents assembly of V0/V1 in the ER or in downstream compartments. It may be that the interaction between Vma21p and V0 is highly dynamic, moving rapidly between bound and unbound states. In the ER, Vma21p is abundant and Vma21p/V0 complexes could be favored, to the exclusion of V0/V1 complexes. In contrast, the concentration of Vma21p molecules in the Golgi is low, and dissociation of Vma21p/V0 complexes could result in a greater population of free V0 sectors that are then competent to assemble with the V1 sector. Once formed, V0/V1 complexes could be relatively stable and not perturbed by the presence of Vma21p.

Vma21p Homologues

Given the crucial role that Vma21p plays in biogenesis of the V0 sector in S. cerevisiae, one would expect to find functional counterparts of Vma21p in other organisms that rely on V-ATPases to carry out fundamental cellular processes. An extensive group of small hydrophobic proteins of molecular mass 9.2-9.4 kDa have been identified that are similar in primary sequence to Vma21p and that are predicted to span the membrane twice. Vma21p-like proteins have been identified in the genomes of all eukaryotic organisms sequenced to date. Yeast Vma21p (NP_011619) shows ~21-25% identity to proteins in Arabidopsis (NP_565728), C. elegans (CAA83627), Drosophila (AAF51611), and humans (XP_377060). The Vma21 proteins from fungi and plants have a carboxy-terminal dilysine motif, suggesting they are ER localized and may function in V-ATPase assembly similar to the yeast Vma21p. Unlike the Vma21 proteins from fungi and plants, the metazoan Vma21 proteins lack a carboxy-terminal dilysine and instead have a single lysine at the -3 position and terminate with an acidic aspartate residue (XKXD). Further analysis of the metazoan homologues may provide insights into whether they are ER localized and function in V-ATPase assembly.

Acknowledgments

This work was supported by the Howard Hughes Medical Institute and by grants from the National Institutes of Health (GM26755 and GM38006) to R.S. and T.H.S., respectively.

Notes

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04-06-0514. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04-06-0514.

Abbreviations used: COP, coat protein complex; CPY, carboxypeptidase Y; gpα-factor, glycosylated pro-α-factor; SIC, semi-intact cell; V-ATPase, vacuolar-type proton-translocating ATPase; VMA, vacuolar membrane ATPase.

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