©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
VMA8 Encodes a 32-kDa V Subunit of the Saccharomyces cerevisiae Vacuolar H-ATPase Required for Function and Assembly of the Enzyme Complex (*)

Laurie A. Graham , Kathryn J. Hill , Tom H. Stevens (§)

From the (1)Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The isolated Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of at least 10 subunits. We have identified VMA8, the gene encoding the 32-kDa subunit of the V-ATPase, by 100% match between the sequences of tryptic peptides and the predicted protein sequence of ORF11. The VMA8 gene contains a 768-base pair open reading frame encoding a 256-amino acid protein with a predicted molecular mass of 29,176 Da. Disruption of VMA8 resulted in a mutant exhibiting pH-sensitive growth, slowed growth under all conditions, and an inability to grow on nonfermentable carbon sources. Vacuolar membranes isolated from vma8 yeast cells exhibited no V-ATPase activity. Immunoblot analysis of vma8 cells revealed normal levels of both V and V subunits. Whereas the V subunits failed to associate with the vacuolar membrane in vma8 cells, the V polypeptides were transported to and stable in the vacuolar membrane. Density gradient fractionation revealed that Vma8p associated only with the fully assembled V-ATPase and did not associate with a separate lower density V subcomplex fraction. Finally, Vma8p was unable to assemble onto the vacuolar membranes in the absence of other V subunits.


INTRODUCTION

The Saccharomyces cerevisiae vacuolar H-translocating ATPase (V-ATPase)()belongs to a family of multisubunit proton-translocating V-type ATPases present in all eukaryotic cells and various bacteria. Members of the V-type ATPase family are necessary for the acidification of several organelles including endosomes, Golgi, secretory vesicles, vacuoles, and lysosomes (1). The proton gradient generated by the ATP hydrolyzing activity of the yeast V-ATPase is utilized by other transporters to drive the accumulation of ions, amino acids, and metabolites into the vacuole(2, 3) .

The V-ATPase is similar in structure and subunit composition to the well characterized FF-ATPase of the mitochondrial inner membrane(4) . The V-ATPase is composed of a V catalytic sector of peripherally associated proteins facing the cytosol that are assembled on the V sector of integral proteins forming the proton-translocating pore. Unlike the FF-ATPase, the V-ATPase is only capable of hydrolyzing ATP coupled to the translocation of protons into the vacuole resulting in vacuolar acidification.

Biochemical analysis has identified at least ten subunits of the yeast V-ATPase ranging in molecular mass from 14 to 100 kDa. The V sector appears to be composed of multiple copies of a 17-kDa proteolipid forming the proton pore in addition to the 36- and 100-kDa subunits(5, 6, 7, 8, 9) . The V sector contains polypeptides of 69, 60, 54, 42, 32, and 27 kDa(10, 11, 12, 13) . The 14-kDa subunit is unique because it behaves biochemically as a V subunit, yet cells lacking this protein fail to transport V subunits to the vacuolar membrane(14) . The 69- and 60-kDa proteins are the catalytic subunits of the V sector, analogous to the and subunits of the FF-type ATPase complex. The remaining subunits, however, share no significant sequence similarity to the , , and FF-type ATPase subunits.

Mutations in a number of vacuolar membrane ATPase (VMA) genes (with the exception of VPH1 encoding the 100-kDa subunit; Refs. 9 and 15) result in a characteristic set of growth phenotypes in yeast. Cells in which a VMA gene has been disrupted exhibit increased sensitivity to calcium and the inability to grow on nonfermentable carbon sources or in media buffered to neutral pH. In addition, vacuolar membranes isolated from these vma mutants lack ATPase activity. Besides the VMA genes that encode V-ATPase subunits, other proteins have been identified that are not associated with the final V-ATPase complex but are required for its assembly (Vma12p, Vma21p, and Vma22p; Refs. 16 and 17).()Loss of any of these nonsubunit proteins also results in a Vma phenotype.

Complete assembly of the V-ATPase requires the presence of every subunit except the 54-kDa subunit encoded by the VMA13 gene (11). Cells lacking any V subunit (e.g. 60-kDa subunit encoded by VMA2) fail to assemble the remaining V subunits onto the vacuolar membrane, but these unassembled proteins remain stable in the cytoplasm(9) . However, these mutants have been found to assemble the V polypeptides and transport them to the vacuole(7) . In contrast, disruption of a gene encoding a V subunit, such as the 17-kDa proteolipid (VMA3) integral membrane protein, results in a significant decrease in the steady-state level of the remaining V subunits, reflecting increased turnover of the unassembled subunits (7).()Although, the loss of a V subunit has no effect on the steady-state level of the V subunits, these peripheral polypeptides are unable to assemble onto the vacuolar membrane.

This paper describes the cloning of VMA8 and the characterization of the encoded 32-kDa polypeptide (Vma8p). Vma8p is a subunit of the V-ATPase and a component of the V complex. Loss of Vma8p from the cell prevents assembly of the remaining V subunits onto the vacuolar membrane. Vma8p shares sequence identity with V-ATPase subunits from a variety of organisms but lacks similarity to any FF-ATPase subunits, suggesting that Vma8p is unique to the V-ATPases.


EXPERIMENTAL PROCEDURES

Strains and Culture Conditions

Yeast strains used in this study are listed in . The wild-type strain is SF838-1D. vma2 (SF838-1DV1) is isogenic to SF838-1D except vma2::LEU2; vma8 (KHY104) is isogenic to SF838-1D except vma8::LEU2. Yeast were grown in YEPD buffered to either pH 5.0 or 7.5 as described by Yamashiro et al. (18) or in S.D. (0.67% yeast nitrogen base, 2% dextrose) supplemented with the appropriate amino acids.

Cloning VMA8

Oligonucleotides synthesized complementary to the 5`-flanking (5`-TTGCCTTTGCCCGTGCCTTA-3`) and 3`-flanking (5`-GCTACCATTGGCGTGGTCTC-3`) sequences of VMA8 were used to amplify by PCR from genomic DNA a 2-kb DNA fragment containing the VMA8 gene. The PCR fragment was digested with the enzymes SstI and BglII to generate a 1837-bp restriction fragment containing VMA8, which was then cloned into the BamHI/SacI sites of the vector pBluescript KS+ (Stratagene, La Jolla, CA) to generate the plasmid pKH3201.

Construction of Disruption Strain

The plasmid pKH3201 was digested with the restriction enzyme BbsI, removing a 700-bp internal VMA8 fragment representing 92% of the open reading frame. A 2.2-kb HpaI LEU2 fragment was subcloned into the blunted BbsI sites of pKH3201 creating vma8::LEU2 (pKH3202). A subclone in which the LEU2 open reading frame was cloned in the opposite orientation to the VMA8 reading frame was chosen for the construction of a null allele. The genomic locus of VMA8 was disrupted by the method of Rothstein(19) . Yeast haploid strain, SF838-1D, was transformed using the lithium acetate method (20) with a 3.3-kb SacI/PstI linear fragment containing vma8::LEU2. Leucine prototrophic transformants were screened by PCR amplification to confirm integration into the correct genomic locus using oligonucleotide primers complementary to genomic sequence flanking the VMA8 open reading frame (5`-GCATCTGTAGTACATAGGTTCCTAACA-3` and 5`-GGCTTACATATTTTTGAAAAGGGTCTT-3`). Correct integration of vma8::LEU2 into the VMA8 genomic locus resulted in the amplification of a 2.3-kb fragment versus a 860-bp wild-type fragment.

Protein Preparation, SDS-PAGE, and Western Blot Analysis

Whole cell extracts and vacuolar membranes were prepared from wild-type, vma2, and vma8 cells for SDS-polyacrylamide gel electrophoresis and immunoblot analysis as described previously(14) .

Proteolipid Extraction

Vacuolar membranes from wild-type and vma8 cells were extracted with chloroform/methanol as described by Ho et al.(11) . Samples were incubated for 30 min at 37 °C in 10 mM Tris, pH 7.4, containing 1 mM EDTA, 8 M urea, 5% SDS, and 5% -mercaptoethanol. Proteins were separated on a 15% SDS-polyacrylamide gel and detected by silver staining the gel.

Epitope Tagging of Vma8p

A 1.9-kb SacI/HindIII fragment containing the VMA8 gene from pKH3201 was subcloned into the centromeric vector pRS316 (21) to create pKH3203. An oligonucleotide duplex encoding a single epitope (YPYDVPDYA) of the influenza virus hemagglutinin protein (HA; Ref. 22) flanked by BglII and BamHI staggered ends was ligated into the BclI restriction site of VMA8 to generate pKH3204 (VMA8::HA). The HA epitope was introduced 8 amino acids from the 3`-terminal stop codon, introducing an additional 1509 Da to the molecular mass of Vma8p. A NdeI restriction enzyme site engineered near the 5` termini of the HA epitope allowed confirmation of the presence of the HA tag. Sequencing of double-stranded template prepared from pKH3204, using the oligonucleotides 5`-GCATCTGTAGTACATAGGTTCCTAACA-3` and 5`-GGCTTACATATTTTTGAAAAGGGTCTT-3`, confirmed that the HA tag was in frame with the VMA8 open reading frame. Whole cell extracts were prepared from vma8 cells (KHY104) carrying pKH3204 and screened by immunoblot analysis using HA monoclonal antibody 12CA5 (Babco, Inc., Berkeley, CA) for expression of the HA tag.

Purification of the Vacuolar ATPase Complex

Vacuolar membranes were isolated from wild-type (SF838-1D) and vma8 (KHY104) yeast cells transformed with either pKH3203 (VMA8) or pKH3204 (VMA8::HA) as described previously(23) . Vacuolar membranes from wild-type or vma8 cells expressing Vma8p-HA were washed with 10 mM Tris, pH 7.4, 1 mM EDTA, solubilized with 2% zwitterionic detergent ZW3-14 (Calbiochem, San Diego, CA), and separated by centrifugation (20 h at 110,000 g) through a 12-35% glycerol density gradient(24, 25) . Fractions were collected in 750-µl aliquots, and a portion of each fraction (100 µl) was assayed immediately for both ATPase and dipeptidyl aminopeptidase B activity(23) . Proteins in the gradient fractions were precipitated with 10% trichloroacetic acid, and samples were prepared for SDS-PAGE as described(24) . Immunoblot analysis of the gradient fractions was performed by probing with antibodies specific for the 100-, 69-, and 36-kDa V-ATPase subunits and Vma8p-HA. Proteins were visualized by chemiluminescence (DuPont NEN) after incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Corp.).

Alkaline Carbonate Extraction of Vacuolar Membranes

Vacuolar membranes from vma8 cells carrying pKH3204 (VMA8::HA) were incubated with 100 mM NaCO, pH 11.5, or 5 M urea as described previously(7) . Pellet and supernatant fractions were analyzed using antibodies specific for the 100-, 69-, 60-, and 36-kDa V-ATPase subunits or Vma8p-HA.

Nitrate Extraction of Vacuolar Membranes

Vacuolar membranes from vma8 cells carrying pKH3204 (VMA8::HA) were treated with potassium nitrate in the presence or absence of 5 mM MgClATP as described previously(24) . Pellet and supernatant fractions from 5 µg of treated vacuolar membranes were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies specific for 100-, 69-, and 36-kDa V-ATPase subunits and Vma8p-HA.

Other Methods

DNA sequencing was performed at the Molecular Genetics Instrumentation Facility, University of Georgia. Protein alignment was generated using the MegAlign sequence analysis program (DNASTAR, Inc.).


RESULTS

Identification of the Gene Encoding the 32-kDa V-ATPase Subunit

The vacuolar ATPase was isolated by glycerol density gradient separation of detergent-solubilized vacuolar membranes. Proteins present in the glycerol gradient fraction possessing the highest ATPase activity were examined by SDS-PAGE. Coomassie staining of this V-ATPase fraction revealed at least 10 major proteins, enriched from the total proteins present in the vacuolar membranes (Fig. 1, right panel). Most of the proteins present in this peak fraction have been identified by various immunological, biochemical, and genetic approaches as subunits of the yeast V-ATPase, with the exception of the 32-kDa protein.


Figure 1: Identification of the 32-kDa subunit of the vacuolar ATPase. Coomassie-stained gels of vacuoles and purified V-ATPase are shown. 10 µg of wild-type vacuolar membrane protein and 3 µg of glycerol gradient isolated V-ATPase were separated on a 8-15% SDS-polyacrylamide gradient gel, and the proteins were visualized by staining with Coomassie Brilliant Blue R-250. Apparent molecular masses of V-ATPase subunits are shown (in kDa). The arrow indicates the 32-kDa product of the VMA8 gene. The asterisk denotes an unidentified protein band.



The SDS-PAGE-separated proteins of the peak V-ATPase fraction were transferred to nitrocellulose, and the individual proteins were visualized by staining with Ponceau S(7) . The band with apparent molecular mass of 32-kDa was excised and subjected to tryptic digestion, the fragments were separated by microbore high performance liquid chromatography, and several well resolved peptides were subject to microsequencing. The sequence of the peptide fragments matched 100% with the sequence available in the GenBank data base (accession number L10830, identified in S. cerevisiae as ORF11 on chromosome V) described as a yeast hypothetical 29.2-kDa protein (Fig. 2A). The open reading frame ORF11, with the predicted amino acid sequence matching the sequence generated from the 32-kDa protein, was located in the AFG1-CYC7 intergenic region of chromosome V. The sequence contained a 768-bp open reading frame encoding a protein of 256 amino acids with a predicted molecular mass of 29,176 Da (Fig. 2A). We have designated this open reading frame as VMA8, the gene encoding a 32-kDa vacuolar membrane ATPase subunit. During the preparation of this manuscript, the VMA8 sequence was reported by Nelson et al.(26) . In that preliminary communication, Nelson et al. described only that vma8 cells exhibited pH-sensitive growth.


Figure 2: Predicted protein sequence of VMA8 and construction of gene disruption. A, translation of VMA8 open reading frame. The sequence of tryptic peptide fragments generated from isolated 32-kDa V-ATPase subunit is shown underlinedabove the Vma8p sequence. The arrow indicates the site of insertion of the HA epitope tag. B, physical map and construction of the gene disruption. The solid line indicates the genomic sequence. The shaded box indicates the VMA8 open reading frame. The solid triangle indicates the approximate site of insertion of the sequence encoding the single HA epitope into the BclI enzyme restriction site. The black box indicates the 2.2-kb HpaI LEU2 fragment used to replace the BbsI fragment within the coding region of VMA8. The half arrows labeled 1 and 2 represent oligonucleotide primers used to PCR amplify the VMA8 fragment. The figure is not drawn to scale.



Cloning and Disruption of VMA8

Utilizing DNA sequence information available through the data base, oligonucleotides were synthesized complementary to the genomic sequence surrounding the VMA8 open reading frame. PCR amplification and subsequent subcloning generated a 1837-bp fragment containing the VMA8 gene (Fig. 2B), which was sufficient for complementation of a genomic disruption strain. The sequence of the open reading frame of the PCR-generated VMA8 was confirmed by double-stranded sequencing.

A disruption allele was constructed by replacement of the VMA8 open reading frame with the LEU2 gene (Fig. 2B). Replacement of VMA8 with vma8::LEU2 at the genomic locus resulted in a mutant displaying growth phenotypes characteristic of other vma mutants. Cells that carried vma8::LEU2 (KHY104) exhibited pH-sensitive growth, the ability to grow in media buffered to pH 5.0 but not to 7.5. The vma8::LEU2 cells also showed increased doubling time in all growth media tested and were incapable of growing on glycerol, a nonfermentable carbon source, even in media buffered to pH 5.0. Vacuolar membranes isolated from vma8 cells completely lacked ATPase activity (<1% wild-type activity). Transformation of vma8 disruption strain with a centromeric plasmid-borne VMA8 PCR-derived gene fragment (pKH3203) resulted in complete complementation of the Vma phenotypes as determined by restoration of wild-type growth and V-ATPase activity (data not shown).

A search of the protein data bases revealed several proteins that demonstrated homology to the yeast 32-kDa protein (Fig. 3). The highest homology was shared with a hypothetical 28.8-kDa protein from Caenorhabditis elegans identified as a putative open reading frame in the genome (GenBank accession number Z27080). The yeast and C. elegans predicted proteins shared 52% identity and 68% similarity as calculated over the entire amino acid sequence. The function of the C. elegans 28-kDa protein is unknown, but its high identity to a yeast V-ATPase subunit suggests that it plays a similar role in C. elegans. As pointed out by Nelson et al.(26) , yeast Vma8p shares 55% identity with a bovine 34-kDa predicted V-ATPase polypeptide subunit. The yeast 32-kDa protein also shared homology (27% identity and 48% similarity) with the NtpD protein subunit of Enterococcus hirae vacuolar type Na-translocating ATPase (27) and with the subunit of the archebacterium Sulfolobus acidocaldarius ATPase (23% identity and 47% similarity, Ref. 28). The suggestion that ORF11 encoded the 32-kDa yeast V-ATPase subunit was first proposed by Takase et al. in their work describing the sequence of the entire ntp operon encoding the subunits of the E. hirae Na-translocating ATPase(27) .


Figure 3: Alignment of Vma8p with homologous protein sequences. Clustal alignment of the predicted open reading frame from S. cerevisiae Vma8p V-ATPase subunit (Sc Vam8p) with the predicted protein sequences from the 28.8-kDa C. elegans hypothetical protein (Ce 28kDa), the E. hirae Na-ATPase NtpD protein (Eh NtpD), and the S. acidocaldarius ATPase subunit (Sa gamma). The sequences that are identical to Vma8p are boxed.



Fate of V-ATPase Subunits in vma8 Cells

In order to determine the effect of the loss of Vma8p on the remaining V-ATPase subunits, we examined the steady-state levels of proteins in whole cell extracts and in isolated vacuolar membranes prepared from wild-type (SF838-1D) and vma8 cells (KHY104). The samples were separated by SDS-PAGE, and the relative protein levels in wild-type and vma8 cells were compared by immunoblot analysis using antibodies specific to each of the subunits. The steady-state protein levels of the 100-, 69-, 60-, 54-, 42-, 36-, and 27-kDa subunits present in whole cell extracts prepared from vma8 cells were indistinguishable from the levels of these proteins present in wild-type cells suggesting that the loss of Vma8p had no effect on the synthesis or stability of the subunits examined (Fig. 4A, left panel).


Figure 4: Comparison of V-ATPase subunit levels in whole cell extracts and vacuolar membranes prepared from wild-type and vma8 cells. A, detection of V-ATPase subunits by immunoblot analysis. 30 µg of whole cell extract protein or 4 µg of vacuolar membrane protein were loaded per lane and then separated by electrophoresis, and immunoblots were probed with antibodies specific for the individual subunits, as described under ``Experimental Procedures.'' B, detection of the 17-kDa proteolipid V-ATPase subunit. Proteins extracted from 10 µg of vacuolar membranes with chloroform/methanol as described under ``Experimental Procedures'' were separated by SDS-PAGE on a 15% acrylamide gel and detected by silver staining.



In contrast to whole cell extracts, vacuolar membranes isolated from vma8 cells (KHY104) contained only two of the seven subunits that could be detected by immunoblot analysis using available antibodies. Only the 100- and 36-kDa subunits could be detected in vacuolar membranes from vma8 cells, but all subunits could be detected in the vacuolar membranes prepared from wild-type cells (Fig. 4A, right panel). Additionally, the 17-kDa vacuolar ATPase proteolipid subunit was present at wild-type levels in the vacuolar membranes of the vma8 mutant as determined by silver-stained chloroform/methanol-extracted proteins (Fig. 4B). The results indicate that the three well-characterized polypeptides of the V sector are all present in the vacuolar membranes of vma8 cells.

In the absence of Vma8p, the 69-, 60-, 54-, 42-, and 27-kDa proteins were not associated with the vacuolar membrane in a vma8 mutant (Fig. 4, right panel), even though they are present in the cell at wild-type levels. Because these proteins are identified as subunits of the ATP-hydrolyzing V sector of the vacuolar ATPase, the loss of Vma8p must prevent the association of these V subunits with the V polypeptides on the vacuolar membrane. Therefore, these data are consistent with the suggestion that Vma8p functions as a subunit of the V subcomplex or is required for its assembly.

Vma8p Is a Subunit of the V-ATPase

To localize Vma8p in the cell and determine if it associates with the V-ATPase, an epitope-tagged version of the protein was generated (see ``Experimental Procedures''). The sequence corresponding to the HA epitope (YPYDVPDYA) was inserted between amino acid Ala and Asp. VMA8::HA complemented the growth phenotypes of the vma8 yeast cells. Vacuolar membranes were isolated from the disruption strain, vma8, carrying either VMA8 or VMA8::HA, and the ATPase activities of the vacuolar membranes were compared. The specific activity of vacuolar membranes from vma8 cells expressing Vma8p-HA was 1.4 µmol of ATP hydrolyzed per min/mg of protein, compared with the specific activity of vma8 expressing Vma8p, which was 1.8 µmol ATP hydrolyzed per min/mg of protein. The V-ATPase containing the epitope-tagged Vma8p possessed approximately 78% of the ATP-hydrolyzing activity of cells carrying the untagged protein and indicated that the introduction of the HA epitope at the 3` terminus of Vma8p did not appear to significantly alter its function in the cell.

Vacuolar membranes isolated from vma8 cells expressing Vma8p-HA were solubilized and separated by centrifugation through a 12-35% glycerol density gradient. Fractions were assayed for both ATPase activity and dipeptidyl aminopeptidase B activity, and immunoblot analysis was performed with antibodies specific to V-ATPase subunits (Fig. 5). The peak ATPase activity was found in fractions 7-9 of the 16 gradient fractions collected, and dipeptidyl aminopeptidase B activity was highest in fraction 4. Dipeptidyl aminopeptidase B is a 120-kDa integral membrane protein component of vacuolar membranes (29) and is fractionated away from the V-ATPase under these conditions as indicated by a separate and distinct dipeptidyl aminopeptidase B activity peak (Fig. 5).


Figure 5: Detection of Vma8p in solubilized vacuolar membranes fractionated on a glycerol density gradient. Vacuolar membranes were isolated from vma8 cells carrying plasmid pKH3204. The proteins were solubilized in 2% ZW3-14 and separated by centrifugation through a 12-35% glycerol density gradient. Protein samples from each fraction were separated by SDS-PAGE and probed with antibodies specific for the 100-, 69-, and 36-kDa V-ATPase subunits and the HA epitope. The arrows indicate fractions possessing peak ATPase and dipeptidyl aminopeptidase B (DPAP B) activity.



The glycerol gradient fractions of solubilized vma8 vacuolar membranes expressing Vma8p-HA were probed with antibodies against two V subunits, the 100- and 36-kDa proteins, and the V 69-kDa catalytic subunit. Immunoblot analysis revealed all three of the V-ATPase subunits colocalized to gradient fractions 7-9, and this corresponded to the fractions possessing the highest ATPase activity, indicating that these fractions contained assembled and functional V-ATPase. Glycerol gradient fractions probed for the epitope-tagged Vma8p showed the protein cofractionating with the active V-ATPase fractions, indicating that Vma8p is a subunit of the vacuolar ATPase. The V 100- and 36-kDa subunits typically showed a biphasic distribution in the glycerol density gradients, results consistent with those previously reported(7, 24) . The high density peak displayed significant cofractionation between the 36- and 100-kDa proteins and ATPase activity and a second, less dense protein peak possibly representing isolated V complex without the associated V subunits.

Fate of Vma8p When VAssembly Is Blocked

The assembly of a functional V-ATPase is dependent on the presence of all the subunits of the complex, with the exception of the 54-kDa subunit (VMA13; Ref. 11). In mutants lacking a subunit of the V complex, such as the 60-kDa protein encoded by the VMA2 gene, the V complex was assembled and present on the vacuolar membrane, but the V subunits remained unassembled and in the cytoplasm of the cell. To determine whether Vma8p behaved like other V subunits, whole cell extracts and vacuolar membranes were prepared from wild-type and vma2 mutant cells expressing Vma8p-HA. Immunoblot analysis of the protein samples was performed by probing with antibodies specific to the 100- and 36-kDa Vsubunits, the 69-kDa catalytic V subunit, and Vma8p-HA. Both the V and V subunits were present in vma2 mutant cells at wild-type levels as shown in the left panels of Fig. 6.


Figure 6: Partitioning of vacuolar ATPase subunits in vma2 cells. Whole cell extracts and vacuolar membrane proteins prepared from wild-type and vma2 cells expressing Vma8p-HA were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against 100-, 69-, and 36-kDa V-ATPase subunits and Vma8p. 4 µg of vacuolar membrane proteins or 30 µg of whole cell proteins were loaded per lane.



As expected, loss of the 60-kDa V protein (encoded by VMA2) prevented the assembly of the V complex and thus the V-ATPase complex, as confirmed by the absence of the 69-kDa protein on the vacuolar membranes of the vma2 cells. The level of the Vma8p-HA detected in whole cell extracts of vma2 cells was identical to that present in wild-type cells expressing Vma8p-HA. The loss of Vma2p had a very dramatic effect on the level of Vma8p associated with the vacuolar membranes without effecting the level present in the cell, as shown by comparing the bottom panels of Fig. 6(labeled Vma8p). A faint signal was detected in the vacuolar membranes of vma2 cells expressing Vma8p-HA but was present at a significantly diminished level compared with Vma8p associated with wild-type vacuolar membranes. Therefore, the behavior of Vma8p in vma2 cells was consistent with the assignment of Vma8p as a subunit of the V sector.

Vma8p Is a Peripheral Membrane Component of the VSector

To determine the nature of the association of Vma8p with membranes, vacuolar membranes were treated with alkaline carbonate or urea. Treated membranes were separated from soluble proteins by centrifugation at high speed (100,000 g). Immunoblot analysis of the pellet and supernatant fractions was performed by probing with antibodies specific to the 100- and 36-kDa V subunits, the 69- and 60-kDa V subunits, and Vma8p-HA (Fig. 7). For vacuolar membranes treated with buffer only, all of the V-ATPase subunits examined were associated with the membrane pellet fraction. A significant fraction of the Vma8p was removed from the vacuolar membrane by treatment with alkaline carbonate or urea. In contrast, the treatment of vacuolar membranes with either alkaline carbonate or 5 M urea did not alter the distribution of the integral 100-kDa V subunit(4, 8) . The protein band below the 100-kDa (identified by an asterisk in Fig. 7) represents the 75-kDa degradation product that remains associated with the membrane fraction (9). In these same samples, the 36-kDa protein was very efficiently released from the vacuolar membrane by these treatments, confirming previously published results characterizing the unique biochemical behavior of this peripheral V subunit(7) . Finally, the 69- and 60-kDa V proteins, forming the catalytic core of the V-ATPase, were largely released into the supernatant fraction following treatment with alkaline carbonate or urea. Like the hydrophilic 60-kDa V-ATPase subunit, Vma8p was not completely extracted from the membrane fraction.


Figure 7: Alkaline sodium carbonate or urea treatment of vacuolar membranes from cells expressing Vma8p-HA. Vacuolar membranes were incubated with 10 mM Tris, pH 7.4, 1 mM EDTA (TE Buffer), 100 mM NaCO, or 5 M urea as described under ``Experimental Procedures.'' The pellet (P) and supernatant (S) fractions from 5 µg of vacuolar membranes were separated by SDS-PAGE and analyzed by probing immunoblots with antibodies specific to the 100-, 69-, 60-, and 36-kDa V-ATPase subunits and Vma8p-HA. The asterisk indicates the 100-kDa degradation product.



The possible association of Vma8p with the V sector was further examined through the use of the chaotropic reagent KNO. Treatment of vacuolar membranes with relatively low concentrations of KNO (100 mM) has been shown to release the 69-, 60-, and 42-kDa V subunits from the membrane in a MgATP-dependent manner(24, 30, 31) . Vacuolar membranes were isolated from cells expressing Vma8p-HA and subjected to KNO treatment in both the presence and the absence of MgATP. The membrane samples were treated with buffer alone, 100 mM KNO, or 100 mM KNO plus 5 mM MgATP and centrifuged at high speed generating a membrane pellet and supernatant fraction. Immunoblot analysis of the samples was performed using antibodies against the 100- and 36-kDa V subunits, the 69-kDa V subunit, and Vma8p-HA. Buffer or 100 mM KNO treatment alone resulted in the release of only trace amounts of any V-ATPase polypeptides into the supernatant (Fig. 8). Treatment with KNO plus MgATP resulted in the release of the 69-kDa protein and Vma8p-HA from the membrane. Neither the 100- or 36-kDa V proteins were removed from the vacuolar membranes under these conditions.


Figure 8: Nitrate extraction of vacuolar membranes from cells expressing Vma8p-HA. Vacuolar membranes were incubated with buffer, 100 mM KNO, or 100 mM KNO, plus 5 mM MgClATP. Pellet (P) and supernatant (S) fractions from 5 µg of vacuolar membranes were separated by SDS-PAGE and analyzed by probing immunoblots with antibodies specific to the 100-, 69-, and 36-kDa V-ATPase subunits and Vma8p-HA.




DISCUSSION

The V-ATPase isolated from yeast is composed of at least ten proteins, ranging in molecular mass from 14 to 100 kDa. A large collection of genes has been identified that encode subunits or nonsubunit proteins required for the assembly of a functional V-ATPase (). Loss of any one of these proteins, except Vph1p (8) or its isoform Stv1p(15) , results in a mutant cell exhibiting a pH-sensitive growth phenotype. A number of uncharacterized vma mutants exist in our laboratory representing mutations in genes possibly encoding additional subunits or assembly factors.

We have cloned and characterized the VMA8 gene encoding the yeast 32-kDa V-ATPase subunit. Peptide sequence generated from the 32-kDa polypeptide associated with the yeast V-ATPase (Vma8p) positively identified the S. cerevisiae ORF11 as an additional VMA gene. Disruption of the VMA8 gene in yeast results in the complete loss of V-ATPase activity. The absence of Vma8p did not affect the assembly of the V sector as determined by the presence of the 100-, 36-, and 17-kDa proteins on vacuolar membranes of vma8 cells, but the V subunits could not assemble in the absence of Vma8p. HA-tagged Vma8p copurified with the V-ATPase activity and behaved as a subunit of the V sector of the complex. Therefore, we conclude that the VMA8 gene encodes the yeast 32-kDa V-ATPase subunit, which is required for V-ATPase function.

The yeast Vma8p exhibits sequence similarity to several predicted amino acid sequences available through the GenBank data base, including a putative protein from C. elegans described as similar to a ``membrane-associated ATPase chain'' (GenBank accession number Z27080) and a 25-kDa subunit (encoded by atpG) of the S. acidocaldarius ATPase (Ref. 28; see Fig. 3). The designation of for the S. acidocaldarius subunit refers only to its position in the ATPase operon, after and . No evidence exists that either the C. elegans putative protein or the S. acidocaldarius subunit are functionally related to the well studied FF-ATPase subunit of Escherichia coli responsible for coupling proton translocation to ATP synthesis(32) . No significant identity exists between the predicted amino acid sequences of the yeast Vma8p and the subunit of the yeast FF-ATPase (Atp3p; Ref. 33), suggesting that the 32-kDa subunit is unique to the vacuolar-type ATPases.

Recently published work by Nelson et al.(26) described the cloning and sequencing of a bovine cDNA-encoding subunit D, a 34-kDa protein that copurifies with the catalytic complex of chromaffin granules. Nelson et al.(26) identified the yeast ORF11 as encoding a putative protein possessing 55% amino acid sequence identity to subunit D of the bovine V-ATPase. These workers coincidentally designated ORF11 as VMA8, the gene encoding the yeast equivalent to the bovine V-ATPase subunit D. A data base search by these workers for proteins with sequences similar to the bovine subunit D also identified several protein sequences including the C. elegans open reading frame, hypothesized to encode a protein related to an ``ATPase subunit.'' Nelson et al. proposed that Vma8p, because it has high identity to the bovine subunit D and to the C. elegans -related subunit, is the yeast V-ATPase subunit equivalent to the subunit of the FF-type ATPase. In an apparent contradiction, Nelson et al. also point out that no significant sequence homology exists between subunit D and the subunit of the FF-ATPase. No evidence exists, despite the reasoning offered by these workers, that the function of Vma8p in the V-ATPase complex is similar to the gamma subunit of the FF-type ATPase.

A comparison between the subunits of the E. hirae Na-translocating ATPase, encoded by the ntp gene cluster, and the well characterized yeast V-ATPase identified several analogous proteins shared by the two ATPase complexes(27) . The yeast 32-kDa protein (Vma8p) encoded by VMA8 had been identified as a protein similar to the NtpD subunit of the Na-ATPase present in the membranes of E. hirae (27). The subunit composition of the E. hirae Na-ATPase is very similar to the yeast V-ATPase, suggesting that it is a V-type ATPase. Interestingly, two subunits encoded by the E. hirae Na-ATPase operon, NtpF (14-kDa) and NtpH (7-kDa), lack corresponding sub-units characterized in yeast, suggesting that additional yeast V-ATPase subunits may exist.

An intact Na-ATPase catalytic sector can be isolated by mild EDTA treatment of E. hirae cell membranes(34) . Interestingly, the three major proteins comprising this intact bacterial V subcomplex are the 69- (NtpA), 52- (NtpB), and 29-kDa (NtpD) subunits of the E. hirae Na-ATPase. The two largest proteins from this bacterial complex are analogous to the 69- and 60-kDa yeast V subunits. As already described, the NtpD protein shares high similarity to the yeast 32-kDa protein characterized in this work. The ability to isolate a V subcomplex suggests that a strong physical association exists between these peripheral subunits (69-, 52-, and 29-kDa), stronger than the association to the V sector.

The physical interaction between Vma8p, the vacuolar membrane, and other V-ATPase subunits was examined by KNO treatment. Proteins that were released only after ATP-dependent treatment in the presence of 100 mM KNO reflected a modified membrane association possibly brought about by a conformational change related to either ATP binding or ATP hydrolysis. Vma8p, like the 69-kDa subunit, was sensitive to this ATP-dependent release from the vacuolar membrane, but the 36- and 100-kDa V subunits were insensitive to the ATP-induced conformational change. The specific ability of ATP to affect the release of V subunits reflects directly on the role of the V subunits in forming the ATP hydrolyzing complex. Conversely, the lack of stimulation of release of the V subunits by ATP directly reflects the role of the V subunits, not in hydrolyzing ATP, but in the formation of the proton pore through the membrane. The results provided by the study of the vacuolar-type ATPase from E. hirae suggest a similar and important role for Vma8p in forming the catalytic core of the yeast vacuolar ATPase together with the 69- and 60-kDa subunits.

  
Table: S. cerevisiae strains and plasmids


  
Table: 0p4in Unpublished data.(119)


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM38006, Human Frontier Science Program Organization Grant RG-389/94M (to T. H. S.), and an American Heart Association Postdoctoral Fellowship (to L. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 503-346-5884; Fax: 503-346-4854; E-mail: Stevens@molbio.uoregon.edu.

The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair; kb, kilobase pair(s); HA, the 9-amino acid epitope (YPYDVPDYA) of the influenza virus hemagglutinin protein; Vma8p-HA, Vma8p carrying the HA epitope.

K. J. Hill and T. H. Stevens, unpublished results.

L. A. Graham and T. H. Stevens, unpublished results.


ACKNOWLEDGEMENTS

We thank Margaret Ho, Cynthia Bauerle, and Margaret Lindorfer for assistance with tryptic peptide fragment sequencing and analysis. We also thank Eric Whitters, Nia Bryant, and Dewaine Jackson for critical reading of the manuscript.


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