©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The SaccharomycescerevisiaeVMA10 Is an Intron-containing Gene Encoding a Novel 13-kDa Subunit of Vacuolar H-ATPase (*)

ubica Supeková , Franti&;ek Supek , Nathan Nelson (§)

From the (1) Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The vacuolar H-ATPase (V-ATPase) functions as a primary proton pump that generates an electrochemical gradient of protons across the membranes of several internal organelles. It is composed of distinct catalytic and membrane sectors, each containing several subunits. We identified a protein (M16) that copurifies with the V-ATPase complex from Saccharomyces cerevisiae and appears to be present at multiple copies/enzyme. Amino acid sequencing of its proteolytic products yielded three nonoverlapping peptide sequences matching an unidentified reading frame located on chromosome VIII. Sequence analysis of cDNA encoding M16 revealed that the gene encoding this protein (VMA10) is interrupted by a 162-nucleotide intron that begins after the ATG codon of the initiator methionine. The cDNA encodes an hydrophilic protein of 12,713 Da with a basic isoelectric point of pH 9. A vma10::URA3 null mutant exhibited growth characteristics typical of other vma disruptant mutants in genes encoding subunits of V-ATPase. The null mutant does not grow on medium buffered at pH 7.5. It fails to accumulate quinacrine into its vacuole, and subunits of the catalytic sector are not assembled onto the vacuolar membrane in the absence of M16. A cold inactivation experiment demonstrated that M16 is a subunit of the membrane sector of V-ATPase. M16 exhibits a significant sequence homology with subunit b of F-ATPase membrane sector.


INTRODUCTION

Proton pumps are among the most important primary pumps in biological systems. They provide the universal high energy intermediate (called the protonmotive force) that can drive numerous secondary uptake processes (1, 2) . Vacuolar H-ATPase (V-ATPase)() is one of these proton pumps, and it is present in every known eukaryotic cell. Its main function is to energize the vacuolar system of eukaryotic cells and to provide the protonmotive force necessary for many biochemical and physiological processes. This enzyme is composed of two distinct structures, the catalytic and the membrane sectors, and each is composed of several subunits (1, 2, 3, 4, 5, 6, 7) . Recently it became apparent that the initial number of subunits implicated in the structure and function of V-ATPase was minimal. Biochemical studies have suggested that yeast V-ATPase is composed of at least 10 subunits ranging in molecular mass from 17 to 100 kDa (8, 9) . However, recent studies suggest that the number of subunits that make V-ATPase is much greater. In addition, several gene products may also function in different steps in the biogenesis and assembly of the enzyme or are necessary for its proper activity (10-15). The family of vacuolar H-ATPases is related to the F-ATPases that are present in the chloroplasts and mitochondria of eukaryotic cells and in most, if not all, eubacteria (1, 2) . The catalytic sector of F-ATPases contains five different subunits. Initial biochemical studies of V-ATPase catalytic sector suggested that it contains five different subunits as well. These subunits were denoted as subunits A to E in order of decreasing molecular weight from 6 to 28 kDa (3) . Very recently it was demonstrated that the enzyme's catalytic sector from insects and Saccharomyces cerevisiae contains an additional sixth subunit (10, 11, 16) . The gene (VMA7) encoding this subunit in yeast was cloned and sequenced, and its product (Vma7p) was denoted as subunit F of the catalytic sector. Even though we do not expect to find more subunits for the catalytic sector of V-ATPase, we do anticipate finding more gene products that may regulate the catalytic sector by inhibiting or promoting the ATPase and proton pumping activities of the enzyme.

In contrast to the subunit structure of the catalytic sector that is generally defined, that of the membrane sector of V-ATPase is largely unknown. So far, four different gene products were clearly shown to comprise the yeast V-ATPase membrane sector (17, 18, 19, 20, 21) . The proteolipid is a pivotal subunit of the membrane sector and is most likely involved in the proton translocating activity of the enzyme (17, 18, 22, 23) . This gene product of VMA3 encodes a highly hydrophobic protein that binds dicyclohexylcarbodiimide. The integrity of this and other subunits of the enzymes is judged by their presence in the purified V-ATPase and, more importantly, by their necessity for the activity and/or assembly of the enzyme. This is checked by disrupting genes encoding specific V-ATPase subunits in yeast, resulting in a phenotype that cannot grow at high pH or high or low calcium concentrations in the medium (18, 22, 23, 24) . In addition, a polypeptide can be defined as a subunit of a multisubunit protein complex only if it is present in stoichiometric amounts of at least one unit/enzyme. The other subunits that were defined as part of the membrane sector of the enzymes are Ac39 and Ac115. Ac39 is a single gene product (VMA6) that is hydrophilic in nature but is copurified with the membrane sector (19, 25) . It is not released by cold inactivation from the membranes. Two genes encode Ac115 (VPH1 and STV1), which contains several potential transmembrane helices (20, 21) . The inclusion of these two gene products as genuine subunits of the enzymes is somewhat controversial because inactivation of each of these genes did not give the phenotype resulted from inactivating the genes encoding all the other subunits (20, 21) . However, when the two genes were simultaneously inactivated, the desired phenotype was obtained (21) . Since a homologous cDNA was discovered in mammalian cells (26) , it may be appropriate to consider Ac115 as a genuine subunit of the enzyme. However, we do not think that it participates directly in the mechanism of action of V-ATPases. The membrane sector of V-ATPase may contain additional hydrophobic subunits that participate in the mechanism of proton conduction across the membrane (12, 27, 28, 29, 30, 31, 32) . Alternatively, the proteolipid by itself may conduct the protons by a completely unknown mechanism.

In looking for additional subunits of the membrane sector, we isolated the V-ATPase from yeast vacuoles. We detected one polypeptide with an apparent molecular mass of 16 kDa on SDS gels that appears to have a larger copy number than the other subunits of the V-ATPase. The polypeptide was subjected to proteolytic cleavage and amino acid sequencing. The resulting amino acid sequences identified a DNA sequence in the GenBank without an assigned reading frame. Interruption of this gene (VMA10) yielded the vma10::URA3 mutant that exhibited a similar phenotype to the other null mutants of yeast V-ATPase.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were purchased from Boehringer Mannheim or New England Biolabs. Peroxidase-conjugated protein A and antibodies were obtained from Sigma. Radioactive chemicals and the ECL antibody detection system were from Amersham Corp. Amplitaq DNA polymerase for PCR amplification was purchased from Perkin Elmer-Cetus Instruments.

Methods

Published procedures were used for transformation of yeast cells by lithium acetate treatment (33) , recombinant DNA methods (34) , purification of yeast genomic DNA (35) , protein determination (36) , assaying ATPase and proton uptake activities (37) , and screening libraries (34) . A 5`-STRETCH yeast cDNA library in gt11 was purchased from Clontech. The library was screened using a P-labeled DNA fragment obtained by PCR from yeast genomic DNA. Positive plaques were isolated, and the cDNA inserts were amplified by PCR using gt11 primers. The resulting DNA fragments were cloned in pCRII plasmid (Invitrogen). Both DNA strands of the isolated cDNA were sequenced using oligonucleotide primers. Western blots were performed according to the protocol of the ECL antibody detection system. Samples were denatured by SDS sample buffer (0.1 M Tris, pH 6.8, 2% SDS, 2% 2-mercaptoethanol, 0.05% bromphenol blue, and 10% glycerol) and electrophoresed on 12% polyacrylamide minigels (Bio-Rad) as described previously (27) . Following electrotransfer at 0.5 A for 15 min, the nitrocellulose filters were blocked for 1 h in a solution containing 100 mM NaCl, 100 mM sodium phosphate, pH 7.5, 0.1% Tween 20, and 5% nonfat dry milk. The blocked filters were incubated with antibodies for 1 h at room temperature at dilution of 1-1000 in a similar solution containing 1% dry milk. Following four washes in the same solution, peroxidase-conjugated secondary antibody or protein A was added to the filters. After incubation for 1 h and four washes with the same solution, the nitrocellulose filters were subjected to the ECL amplification procedure. The filters were exposed to Kodak X-Omat AR film for 5-30 s.

Construction of Epitope-tagged Vma10p

A DNA fragment encoding the amino acid sequence: YPYDVPDYAS (tag), which is influenza hemagglutinine epitope (HA), was introduced by PCR into the carboxyl terminus of the reading frame of VMA10 gene. Following cloning into EcoRI and SphI sites of the YPN2 plasmid (22) , the correct insertion was verified by DNA sequencing. Transformation of vma10 null mutants with the plasmid containing the tagged gene resulted in complementation of the null phenotype. Monoclonal antibody against the epitope tag (12CA5 mouse cell line) was purchased from BAbCO and used at a dilution of 1:1000.

Yeast Strains and Analysis of Mutants

S. cerevisiae strain W303-1B was used for this study. Wild-type and mutants were grown in YPD medium containing 1% yeast extract, 2% Bacto-peptone, and 2% dextrose. The medium was buffered by 50 mM Mes and 50 mM Mops, and the pH was adjusted as specified by NaOH (24) . Agar plates were prepared by the addition of 2% agar to the YPD buffer medium at the given pH. After transformation by the lithium acetate method (33) , yeast cells were grown on minimal plates containing 0.67% yeast nitrogen base, 2% dextrose, 0.1% casamino acids, 2% agar, and the appropriate nutritional requirements. For measurements of quinacrine uptake, 1 ml of yeast culture (A = 0.8) was sedimented, resuspended in 0.1 ml of YPD containing 100 mM Hepes, pH 7.6, and 200 µM quinacrine. After incubation at 30 °C for 10 min, the cells were washed 3 times with ice-cold solution containing 100 mM Hepes, pH 7.6, and 2% dextrose. The cells were resuspended in 0.1 ml of the same buffer, mixed with equal volume of 1% low melting point agarose, mounted on glass slides, covered with coverslips, and observed for fluorescence or Nomarski within 10 min. For preparation of vacuoles, cells were grown in YPD medium adjusted to pH 5.5 by HCl and harvested at cell density of about A = 0.8. Vacuolar membranes were prepared according to Uchida et al.(38) , except that the homogenization buffer contained no magnesium and the vacuoles were washed only once with the EDTA buffer. ATP-dependent proton uptake activity was assayed by following the absorbance changes of acridine orange at 490-540 nm as described previously (37) . The 1-ml reaction mixture contained 20 mM Mops-Tris, pH 7, 150 mM KCl, and 15 µM acridine orange. Iso-lated yeast vacuoles containing 5-20 µg of protein were added to the reaction mixture followed by 10 µl of 0.1 M MgATP. The reaction was terminated by the addition of 1 µl of 1 mM carbonyl cyanide p-trifluoromethoxyphenylhydrazone.

Purification of Yeast V-ATPase and Isolation of Proteolitic Cleaved Polypeptides

Yeast cells were grown in 10 20-liter fermentors in YPD medium, and vacuoles were isolated as was described above. V-ATPase (about 0.5 mg of protein) was purified from the isolated vacuoles as described previously (22, 38, 39) . Glycerol gradient fractions containing the purified V-ATPase were precipitated by the addition of 3 volumes of cold ethanol, incubated for 15 min at -20 °C, and centrifuged at 20,000 g for 20 min. The resulting pellet was dissolved in a 1 ml of solution containing 20 mM Mops-NaOH, pH 7.5, 1% SDS, and 0.1% mercaptoethanol. Two 0.5-ml samples were applied onto sucrose gradients (7-35%) in a buffer containing 10 mM Mops-NaOH, pH 7.5, 0.1% SDS, and 0.01% mercaptoethanol. The gradients were centrifuged at 15 °C in a SW60 rotor at 57,000 rpm for 15 h. Ten fractions were collected from each tube and analyzed for subunits content by electrophoresis on 12.5% SDS-polyacrylamide gel. Similar gels were electrotransferred onto nitrocellulose filters that were decorated by the subunit-specific antibodies (Fig. 1). In this way the Coomassie Blue-stained bands were identified as the specified subunits in Fig. 1.


Figure 1: Fractionation of purified V-ATPase on sucrose gradient containing SDS. The purified V-ATPase preparation was processed as described under ``Experimental Procedures'' and applied on top of 3.5-ml sucrose gradients (30%-7%) containing 10 mM Mops-NaOH, pH 7.5, 0.1% SDS, and 0.01% 2-mercaptoethanol. After 15 h of centrifugation at 57,000 rpm in a SW60 rotor, 10 fractions were collected from the bottom and analyzed by SDS-PAGE and immunodecoration with subunit specific antibodies. Top left, SDS gel of the purified V-ATPase stained with Coomassie Blue. The identified subunits are indicated; M115 and M39 are the subunits that were previously denoted as Ac115 and Ac39, respectively. The prominent protein band between subunits E and F is M16. Top right, Coomassie Blue-stained SDS gel of fractions collected from the sucrose gradient. Immunodecoration with antibodies raised against V-ATPase subunits revealed that additional stained bands located between subunits A and E are degradation products of subunit A. The prominent band with apparent M = 16,000 did not cross-react with any of the antibodies used. Bottom right, identification of V-ATPase subunits in the gradient fractions by antibodies raised against individual subunits. Five gels, identical to the stained gel, were electrotransferred to nitrocellulose filters and decorated with the specified subunit-specific antibodies. The position of the individual subunits was matched using prestained molecular weight markers.



To obtain internal amino acid sequence of Vma10p, fractions 6 and 7 were dissociated in sample buffer containing 0.1 M Tris-Cl, pH 6.8, 2% SDS, 2% mercaptoethanol, 0.05% bromphenol blue, and 10% glycerol. The dissociated sample was electrophoresed using Mini-PROTEAN II apparatus (Bio-Rad) on two 15-well 12.5% polyacrylamide gels. One of the gels was briefly stained by Coomassie Blue, destained, and washed with distilled water 3 times, 5 min each time. The band at 16 kDa (above subunit F) was excised and briefly (about 5 min) lyophilized. A 10-well minigel of 15% polyacrylamide was polymerized and incubated overnight at room temperature. About 5 µl of a solution containing 0.1 M Tris-Cl, pH 6.8, 10% glycerol, bromphenol blue, 0.05% SDS, and 0.5 µg of V8 protease (Boehringer Mannheim) was applied into each well and electrophoresed until the stain entered the top of the stacking gel. Then, the slices containing Vma10p were applied into the wells. Electrophoresis was performed at 30 V for 40 min and then at 200 V for an additional 25 min. The proteins were electrobloted onto an Immobilon filter (Millipore) according to Matsudaira (40) . The stained bands were excised and subjected to amino acid sequencing by a gas-phase Applied Biosystems sequenator. The second gel was electrotransferred onto Immobilon filters, the filters were stained with Amido Black (naphthol blue black from Fluka), and the 16-kDa protein band was excised and subjected to trypsin cleavage as described previously. Polypeptides were separated by reverse phase HPLC and sequenced.


RESULTS

Hydrophobic membrane proteins are frequently hard to detect in SDS gels stained with Coomassie Blue. The main obstacle to the isolation of large quantities of such hydrophobic polypeptides is the presence of phospholipids and other hydrophobic substances in the preparation that hamper attempts to concentrate the sample. Concentrating the sample usually results in the loss of resolution on the SDS gel. Therefore, we devised a method for isolating these polypeptides in large quantities (41). The method included the isolation and purification of the membrane protein and precipitation of the proteins by alcohol (see ``Experimental Procedures'' and Fig. 1 ). The resulting pellet was dissolved in a buffered solution containing 1% SDS and 0.1% mercaptoethanol and was subjected to a sucrose gradient centrifugation in a solution containing 0.1% SDS and 0.01% mercaptoethanol. This treatment resulted in the dissociation of polypeptides from the membrane and the displacement of phospholipids by SDS. Therefore, many phospholipids ran as mixed micelles with SDS and appeared at the bottom of the gradient (see Fig. 1, lane1). Polypeptides that were not highly hydrophobic and at low molecular weight ran at the top of the gradients (Fig. 1, lanes5-9). After analyzing the fractions on SDS gels, similar fractions can be combined and reprecipitated with ethanol and rerun on a sucrose gradient as before. Repetition of these gradients will yield a much purer preparation of low molecular weight subunits than in the original preparation. As shown in Fig. 1, we identified several subunits of the enzyme by specific antibodies that were available in our laboratory. Consequently, we were able to identify new proteins. Fig. 1shows that a prominent protein band with an apparent mass of about 16 kDa was present on the gel right above subunit F. This protein was transferred to Immobilon and subjected to trypsin treatment. After separation of the polypeptides by HPLC, the polypeptides were sequenced. In addition, another preparation of this polypeptide was cleaved by V8 protease as described previously (39, 41) . Sequencing of one of the polypeptides resulting from the V8 cleavage gave the amino acid sequence: FEQKNAGGV. Sequencing two of the polypeptides resulting from the trypsin cleavage gave the amino acid sequences NGIATLLQAEK and KAEAGVQGELAEIK.

A search in the GenBank revealed that the amino acid sequences can be correctly translated from sequences within chromosome VIII that were published in the GenBank in May 1994 (U00062) and as a publication in October 1994 (43) . However, there was no assignment for an open reading frame in the published sequence. The translated reading frame identified by the alignment with the above amino acid sequences was situated between the reading frames encoding a protein related to aldehyde dehydrogenase (on the opposite DNA strand) and an open reading frame encoding a protein exhibiting weak homology to the product of HIT1 gene (on the same DNA strand). Since the aforementioned amino acid sequences exhibited 100% identity with the translated DNA fragment from chromosome VIII, we decided to examine whether this DNA fragment encoded the protein that we isolated as a potential subunit of the membrane sector of V-ATPase. Two oligonucleotides were synthesized using the flanking regions of the suspected reading frame as the following: ACC ACA GAA TTC CGC CAT ACT TGC AAA TTG CCA CAC C and TGA AAA GCA TGC TAC TAT TCT GCA AAT ACT ATT ATA. The oligonucleotides contained the respective EcoRI and SphI restriction sites for cloning at the same sites in the YPN2 plasmid (see Fig. 2and Ref. 22). To eliminate the possibility that two identical genes are present in the yeast genome, we performed a Southern blot analysis probed with a P-labeled DNA fragment corresponding to the proposed reading frame of the gene. Southern analysis (not shown) indicated the presence of a single gene in the yeast genome. A separate set of oligonucleotides was designed to disrupt the gene (Fig. 2). The URA3 gene was introduced into the construct by PCR, and the resulting DNA fragment was used for transforming W-303-1b yeast cells. Yeast colonies that grew on minimal plates without uracil were subjected to further analysis. Colonies that did not grow on YPD plates buffered at pH 7.5 were further analyzed by PCR for the correct disruption of the gene. As shown in Fig. 3, the null mutant vma10::URA3 failed to grow at pH 7.5 like any other mutant in which a gene encoding V-ATPase subunit had been interrupted. These results suggested that, indeed, the proposed open reading frame is the gene VMA10 that encodes the protein Vma10p.


Figure 2: Construction of a disrupted allele of the VMA10 gene. A novel HANNH method of gene disruption was utilized for the generation of vma10 null mutation (H. Nelson and N. Nelson submitted for publication). Two oligonucleotides marked in the figure as A and B (for their sequence, see ``Results'') were used for PCR amplification of the VMA10 gene and its flanking regions from yeast chromosomal DNA. The second set of primers (C and D) was designed where the 3` part of the oligonucleotides matches internal sequences of VMA10 and 5` parts of oligonucleotides were derived from the sequences of the URA3 gene. The combinations of primers A + C and B + D were used for amplification of VMA10-flanking regions from the first PCR product with the URA3 gene. The final construct was created in two more PCR steps. First the 5`-flanking region of VMA10 was fused to a URA3 gene, and then the 3`-flanking region of VMA10 was added by amplification with primers A and B. The product of the last PCR contains the URA3 gene in place of VMA10 coding sequence and was used for transformation to create the vma10::URA3 strain. The toppart of the picture shows the position of the initiator methionine (M) and intron (region from M to the start of VMA10) as revealed by cDNA sequencing.




Figure 3: The phenotype of mutants containing a disrupted VMA10 gene. The VMA10 gene was interrupted as described under ``Experimental Procedures'' and in the legend to Fig. 2. Panel A, chromosomal DNAs from wild-type cells W303B and one transformed colony that grew on minimal medium lacking uracil, were amplified by PCR using primers A and B (see Fig. 2). PCR products were analyzed by electrophoresis in 1% agarose gel. Lane 1, molecular weight standards from top to bottom were as follows: 23, 9.4, 6.6, 4.4, 2.3, 2, 1.4, 1.1, 0.9, and 0.6 kilobases; lane 2; wild-type strain; lane 3; vma10::URA3 mutant. Panel B, growth on YPD plates buffered to the indicated pH. 1, W303B; 2, vma10::URA3; 3, vma10::URA3 transformed with the YPN2 plasmid; 4, vma10::URA3 transformed with YPN2 plasmid bearing the VMA10 gene. Each colony in the figure represents an independent transformant.



However, we could not identify the initiator methionine within the proposed reading frame in the published DNA sequence. We therefore concluded that this gene was probably interrupted by an intron and its initiator methionine was upstream of the first amino acid that could be read from translation of the DNA. To prove this point, a yeast cDNA library was screened using a P-labeled DNA fragment encoding the proposed reading frame. The resulting positive plaques were isolated, and the cDNAs were isolated from the gt11 by PCR and cloned by the TA procedure into pCRII plasmid. Sequencing of the cDNA clearly showed that, as shown in Fig. 4, there was an upstream initiator methionine and the reading frame was interrupted by an intron of 162 nucleotides. The mRNA started at nucleotide 243 (Fig. 4) and ended following the polyadenylation signal AATAAA that begins at nucleotide 827. Consequently, the VMA10 gene was expressed as a spliced mRNA. Search in GenBank revealed that Vma10p exhibits 24% identity and about 40% similarity with subunits b of F-ATPases from Escherichia coli or Vibrio alginolyticus (accession number X16050). Amino acid sequence alignment also indicate possible relation to NtpF gene product from the gene cluster for Na-translocating ATPase from Enterococcus hirae(44) . BESTFIT program showed 29% identity and 46% similarity between Vma10p and NtpF.


Figure 4: Nucleotide and derived amino acid sequences of VMA10. The position of the initiator methionine was determined by sequencing of the cDNA isolated from the gt11 library. The amino acid sequences of the peptides are underlined. The cDNA nucleotide sequence has been submitted to GenBank with accession number U21240. The intron starts at nucleotide 260 and ends at nucleotide 421.



To further analyze the phenotype of vma10 null mutation, we followed the quinacrine accumulation both in wild-type and null mutant cells. As shown in Fig. 5the null mutants failed to accumulate quinacrine into their vacuoles. In addition, vacuoles isolated from the null mutant exhibited no ATP-dependent proton uptake activity (Fig. 6). It was demonstrated in our and other laboratories (19, 20, 21, 22, 23, 24) that a null mutation in one of the V-ATPase subunits prevents the assembly of the other subunits in the catalytic sector of V-ATPase. Therefore, we checked the assembly of the different V-ATPase subunits into the vacuole membranes. As shown in Fig. 7, the null mutation vma10::URA3 prevented the assembly of subunits A (Vma1p), C (Vma5p), E (Vma4p), and M39 (Vma6p), the latter of which is a subunit of the membrane sector of the enzyme. In contrast, the null mutation vma10::URA3 cells synthesized similar amounts of these subunits as the wild-type cells (Fig. 4, cell lysate). It is particularly interesting that the membrane sector subunit M39 is also not present in the isolated vacuoles of the mutant cells. This observation is in line with other observations showing that mutations in the catalytic sector subunits did not prevent the assembly of the membrane sector subunits. However, mutations that deleted one of the membrane sector subunits influenced the assembly of the other membrane sector subunits (19, 20, 21, 22) . Vma12p is a vacuolar membrane protein that does not copurify with the V-ATPase (14) . Null mutation in the gene (VMA12) encoding this protein prevented the assembly of the enzyme. The lack of Vma10p in its corresponding null mutant drastically reduced the amount of Vma12p in the vacuolar membranes.


Figure 5: The mutant with a disrupted VMA10 gene does not accumulate quinacrine into its vacuole. Accumulation of quinacrine was measured as described under ``Experimental Procedures.'' Nomarski and fluorescence images are shown. Top, fluorescence picture of vma10 null mutant and the wild-type (WT). Bottom, vma10 null mutant cells as seen using Nomarski optics.




Figure 6: The isolated vacuoles of vma10::URA3 null mutant lack ATP-dependent proton uptake activity. About 20 µg of isolated vacuolar membranes were used for measuring ATP-dependent acidification of membrane vesicles by following a decrease in absorption at 490-540 nm, as described in detail under ``Experimental Procedures.'' Where indicated, 1 µmol of MgATP or 1 nmol of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were added to the 1 ml reaction mixture. Trace1, wild-type membrane vesicles (W303B); trace2, vma10::URA3 mutant membrane vesicles.




Figure 7: Subunits of the catalytic sector are not assembled onto the vacuolar membrane in the absence of Vma10p. Crude cell lysates from wild-type cells and the vma10 null mutant were prepared as described previously (10). Vacuoles were prepared from 5 liters of the corresponding yeast culture grown in YPD, pH 5.5 (38). Fifty µg of cell lysate protein or 5 µg of purified membranes were loaded/lane of 12% SDS-polyacrylamide gel and, after separation, transferred to nitrocellulose membrane. The presence of V-ATPase subunits was detected by affinity-purified antibodies raised against indicated gene products. Lane 1, wild-type cells; lane 2, vma10 null mutant.



Cold inactivation of V-ATPases resulted in inactivation of the enzyme and dissociation of the catalytic sector from the membrane (10, 27, 28, 39) . To assess whether Vma10p is a membrane-associated protein, we performed cold inactivation with the isolated vacuoles from wild-type cells that carried an epitope-tagged Vma10p. The gene encoding the tagged Vma10p complemented the corresponding null mutation, and it grew well on a medium buffered at pH 7.5. Vacuoles were isolated from these cells and subjected to cold inactivation in the presence and absence of MgATP (10, 22, 39) . As shown in Fig. 8, in the absence of MgATP, no loss of V-ATPase subunits from the vacuoles was observed. In contrast, in the presence of MgATP, a major loss of subunits A (Vma1p) and C (Vma5p) into the supernatant was observed. M39 (Vma6p) was also released to the supernatant but to a much lesser extent than the catalytic sector subunits. Fig. 8also shows that cold inactivation in the presence of MgATP failed to release Vma10p from the membrane. These experiments support our conclusion that VMA10 encodes a V-ATPase subunit that is associated with the membrane sector of the enzyme.


Figure 8: Vma10p is not released from vacuolar membrane by cold inactivation. The vma10::URA3 strain was transformed with YPN2 plasmid carrying VMA10 gene containing an epitope tag at its carboxyl terminus. The resulting transformant was grown in 20 liters of YPD medium, and vacuolar membrane vesicles were isolated. Membranes (0.75 mg) were diluted into 3 ml of buffer containing 20 mM Mops-Tris, pH 7.0, 250 mM NaCl, and 1 mM dithiothreitol, and then divided into three parts. MgATP was added to one of them to give 5 mM final concentration. After 2 h of incubation on ice, the sample containing MgATP and one of the samples lacking MgATP were centrifuged at 150,000 g for 25 min. Both supernatants, as well as a sample that was not centrifuged, were concentrated by trichloroacetic acid precipitation and dissolved in 0.1 ml of SDS dissociation buffer. Ten µl of each sample were electrophoresed in 12% polyacrylamide gel, and after transfer to nitrocellulose, membranes were decorated with antibodies against the indicated subunit. Lane 1, starting material of purified vacuoles; lane 2, supernatant after cold treatment without MgATP; lane 3, supernatant after cold treatment in the presence of MgATP.




DISCUSSION

Since the first isolation of V-ATPase from yeast vacuoles (38) the number of reported subunits of this enzyme has increased (8, 10, 41) . This resulted primarily from advancement in techniques used for the purification of the enzyme and more importantly by identification of genes encoding subunits of the enzyme utilizing the specific phenotype resulting from their inactivation (22, 24) . Recently the gene VMA7 was shown to encode subunit F of the catalytic sector of yeast V-ATPase (10, 11) . We believe that all subunits in the catalytic sector have now been identified and that it is composed of six subunits, denoted as subunits A to F in the order of decreasing molecular mass from 69 to 14 kDa. The composition of the membrane sector is far from being settled. So far, only the proteolipid (Vma3p) withstands the criterion for a genuine membrane sector subunit that may be directly involved in proton conduction across the membrane (23) . Since this protein is highly hydrophobic and contains only one charged group inside the membrane, it is likely that other membrane proteins may be involved in proton conduction across the membrane sector of V-ATPase. In addition, it was demonstrated that the membrane sector of V-ATPase provides the template for the assembly of the enzyme (19, 22) . The assembly of the catalytic sector is totally dependent on the proper assembly of the membrane sector, and the sequence of events in the assembly of V-ATPase is that the membrane sector is assembled first followed by assembly of the catalytic sector. These observations lead us to propose that more subunits will be found to comprise the membrane sector of V-ATPase (2, 3) .

In all preparations of purified yeast V-ATPase, we observed a prominent band of approximately 16 kDa. Since dicyclohexylcarbodiimide labeling coincides with this prominent band, we assumed that the band resulted from the presence of the proteolipid at this position. However, analysis with epitope-tagged proteolipid led us to conclude that the Coomassie Brilliant Blue-stained 16 kDa band did not result from the stained proteolipid (not shown). Therefore, we isolated this protein band, subjected it to a proteolytic cleavage and obtained the amino acid sequences of the resulting polypeptides. A search of the GenBank data base with these amino acid sequences identified a potential gene in chromosome VIII that was not previously delineated as an open reading frame. Since we knew that this gene was expressed and the gene product was present in the purified V-ATPase, we looked for the possibility that this gene contained an intron and the initiator methionine was situated somewhere upstream of the reading frame. We were successful in isolating from a cDNA library a full-length clone corresponding to the VMA10 gene containing an initiator methionine that was 162 nucleotides upstream from the nucleotides encoding the second amino acid (serine). The intron starts with the canonical signal of GT and ends with the expected nucleotides AG. Introns are quite rare in yeast, and most of them, as does VMA10, start immediately after the initiator methionine (42).

The integrity of Vma10p as a V-ATPase subunit was checked by all of the available techniques for this purpose. Disruption of the VMA10 gene yielded a mutant with identical phenotype to all the other null mutations in V-ATPase subunits (10, 11, 24, 41) . This phenotype could be complemented by transformation with the plasmid carrying a VMA10 gene. The gene product denoted M16 for membrane protein had an apparent molecular mass of 16 kDa. We would like to suggest that the nomenclature of the other membrane proteins contain the prefix M. This would change Ac39 and Ac115 to M115 and M39. The calculated molecular mass of M16 (Vma10p) was about 13 kDa, and the slower migration on the gel may be a result of its high content of charged amino acids. Even though the predicted amino acid sequence M16 was highly hydrophilic, it behaved like a genuine membrane-associated protein. It was not released from vacuoles by cold inactivation, and the null mutation in this gene prevented not only the assembly of the catalytic sector subunit but also the membrane sector subunit. It is noteworthy that even though the null mutation in the VMA10 gene drastically reduced the assembled amounts of M39 and Vma12p, it did not affect the assembly of the proteolipid into the membrane (not shown). This observation endorses our previous notion that the proteolipid serves as a template for the assembly of remaining subunits in the membrane sector and thereby for the assembly of the catalytic sector (22) . The other criteria that indicate that M16 was a genuine subunit of the membrane sector was its copurification with the enzyme and its presence in stoichiometric amounts with the other subunits of the enzyme. Preliminary results of quantitative amino acid analysis revealed that M16 is present at least as three copies/enzyme (not shown). As with all the other subunits of this enzyme, the precise function of M16 is not known. However, the homology between M16 and subunit b of F-ATPases may suggest similar function in energy coupling and/or assembly of the catalytic sector onto the membrane sector. Very recently, we cloned a cDNA encoding M16 in bovine adrenal medulla.() This finding together with the homology of M16 to bacterial subunit b of F-ATPase, suggests that all V-ATPases including those of archaebacteria contain equivalent subunits to M16.

The discovery of M16 as a subunit of the membrane sector does not lessen the necessity to look for additional hydrophobic protein(s) that may function alongside the proteolipid in proton conduction across the membrane. Indeed, in preparations of V-ATPases from mammalian and yeast sources, such a protein candidate was identified as a protein band that migrates on SDS gels at about 20 kDa (12, 27, 28, 29, 30, 31, 32) . We are actively looking for the genes or cDNAs encoding this polypeptide, which, when found, we suggest to name it VMA9. With the recent discovery of numerous genes encoding subunits and factors that influence the assembly of V-ATPase, this enzyme has become an attractive model for the study of biogenesis and the assembly of complexed membrane proteins.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21240.

§
To whom correspondence should be addressed. Tel.: 201-235-3790; Fax: 201-235-5848.

The abbreviations used are: V-ATPase, vacuolar H-ATPase; PCR, polymerase chain reaction; Mes, 4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid; HPLC, high performance liquid chromatography.

L. Supekova, M. Sbia, F. Supek, and N. Nelson, unpublished results.


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