Yeast Vacuolar Proton-translocating ATPase Research Articles

Genome-Wide Identification of Genes Required for Yeast Growth Under Imatinib Stress: Vacuolar H+-ATPase Function Is an Important Target of This Anticancer Drug

Imatinib is a highly selective tyrosine kinase inhibitor of the oncogenic kinase Bcr-Abl, the result of a chromosomal abnormality that is associated with chronic myeloid leukaemia (CML). Despite the success of this target-directed therapy, imatinib resistance is an emerging problem, especially in advanced stages of CML. In this study, we explored the yeast Saccharomyces cerevisiae as a model eukaryotic system to better understand the mode of action of imatinib, as well as potential mechanisms of resistance to this drug. Using a systematic approach, we screened a yeast haploid deletion collection with individual knockouts of most nonessential yeast genes, and identified 51 genes that are required for yeast resistance to imatinib. The genes identified are involved mainly in DNA repair and transcription control, cell cycle control and differentiation, vacuolar pH homeostasis, vesicular transport, and protein trafficking. Remarkably, approximately 80% of the genes identified in our screen have human orthologs. The vacuolar pH homeostasis function is associated to our dataset by 13 genes that encode subunits and assembly factors of the yeast vacuolar proton-translocating ATPase (V-ATPase). Further studies using fluorescence microscopy showed that physiological acidification of the vacuole is severely compromised following imatinib treatment of yeast cells, an effect that was found to be dose dependent. Results suggest that imatinib might act as an effective inhibitor of V-ATPase function in yeast, identifying V-ATPase activity and vacuolar function as novel imatinib targets.

RAVE Is Essential for the Efficient Assembly of the C Subunit with the Vacuolar H+-ATPase

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.

Identification of a Domain in the Vo Subunit d That Is Critical for Coupling of the Yeast Vacuolar Proton-translocating ATPase

Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V(1)V(o), mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Delta and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.

Repression of Cell-Cell Fusion by Components of the C. elegans Vacuolar ATPase Complex

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.

The Yeast Vacuolar Proton-translocating ATPase Contains a Subunit Homologous to the Manduca sexta and Bovine e Subunits That Is Essential for Function

The yeast cwh36Delta mutant was identified in a screen for yeast mutants exhibiting a Vma(-) phenotype suggestive of loss of vacuolar proton-translocating ATPase (V-ATPase) activity. The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A. We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma(-) growth phenotype of the cwh36Delta mutant. YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase. The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast. Vma9p is localized to the vacuole but fails to reach the vacuole in a mutant lacking one of the integral membrane subunits of the V-ATPase. Vma9p is associated with the yeast V-ATPase complex in vacuolar membranes, as demonstrated by co-immunoprecipitation with known V-ATPase subunits and glycerol gradient fractionation of solubilized vacuolar membranes. Based on this evidence, we propose that Vma9p is a genuine subunit of the yeast V-ATPase and that e subunits may be a functionally essential part of all eukaryotic V-ATPases.

Transmembrane Topography of the 100-kDa a Subunit (Vph1p) of the Yeast Vacuolar Proton-translocating ATPase

The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less mutant, with 12 mutants showing greater than 70% of wild type activity. To evaluate their disposition with respect to the membrane, vacuoles were treated in the presence or absence of the impermeant sulfhydryl reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) followed by the membrane permeable sulfhydryl reagent 3-(N-maleimidylpropionyl) biocytin (MPB). Three of the 12 active cysteine mutants were not labeled by MPB. The mutants E3C, D89C, T161C, S266C, N447C, K450C, and S703C were labeled by MPB in an AMS-protectable manner, suggesting a cytoplasmic orientation, whereas G602C and S840C showed minimal protection by AMS, suggesting a lumenal orientation. Factor Xa cleavage sites were introduced at His-499, Leu-560, and Pro-606. Cleavage at 560 was observed in the absence of detergent, suggesting a cytoplasmic orientation for this site. Based on these results, we propose a model of the a subunit containing nine transmembrane segments, with the amino terminus facing the cytoplasm and the carboxyl terminus facing the lumen.

Assembly of the yeast vacuolar proton-translocating ATPase.

The yeast vacuolar proton-translocating ATPase (V-ATPase) is the best characterized member of the V-ATPase family. Biochemical and genetic screens led to the identification of a large number of genes in yeast, designated VMA, encoding proteins required to assemble a functional V-ATPase. A total of thirteen genes encode subunits of the final enzyme complex. In addition to subunit-encoding genes, we have identified three genes that code for proteins that are not part of the final V-ATPase complex yet required for its assembly. We refer to these nonsubunit Vma proteins as assembly factors, since their function is dedicated to assembling the V-ATPase. The assembly factors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmic reticulum (ER) and aid the assembly of newly synthesized V-ATPase subunits that are translocated into the ER membrane. At least two of these proteins, Vma12p and Vma22p, function together in an assembly complex and interact directly with nascent V-ATPase subunits.

Mutational Analysis of the Nucleotide Binding Sites of the Yeast Vacuolar Proton-translocating ATPase

To further define the structure of the nucleotide binding sites on the vacuolar proton-translocating ATPase (V-ATPase), the role of aromatic residues at the catalytic sites was probed using site-directed mutagenesis of the VMA1 gene that encodes the A subunit in yeast. Substitutions were made at three positions (Phe452, Tyr532, and Phe538) that correspond to residues observed in the crystal structure of the homologous beta subunit of the bovine mitochondrial F-ATPase to be in proximity to the adenine ring of bound ATP. Although conservative substitutions at these positions had relatively little effect on V-ATPase activity, replacement with nonaromatic residues (such as alanine or serine) caused either a complete loss of activity (F452A) or a decrease in the affinity for ATP (Y532S and F538A). The F452A mutation also appeared to reduce stability of the V-ATPase complex. These results suggest that aromatic or hydrophobic residues at these positions are essential to maintain activity and/or high affinity binding to the catalytic sites of the V-ATPase. Site-directed mutations were also made at residues (Phe479 and Arg483) that are postulated to be contributed by the A subunit to the noncatalytic nucleotide binding sites. Generally, substitutions at these positions led to decreases in activity ranging from 30 to 70% relative to wild type as well as modest decreases in Km for ATP. Interestingly, the R483E and R483Q mutants showed a time-dependent increase in ATPase activity following addition of ATP, suggesting that events at the noncatalytic sites may modulate the catalytic activity of the enzyme.

VMA12 Encodes a Yeast Endoplasmic Reticulum Protein Required for Vacuolar H+-ATPase Assembly

The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V0) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa. Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex. One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains. Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER). In cells lacking Vma12p (vma12Delta), the 100-kDa subunit of the V0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1/2 approximately 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12Delta cells. These data indicate that Vma12p functions in the ER after the insertion of V0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V0 subunits into a complex in the ER, and that assembly is required for the stability of the V0 subunits and their transport as a complex out of this compartment.

V1-situated Stalk Subunits of the Yeast Vacuolar Proton-translocating ATPase

The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V1) and an integral membrane sector (V0), each composed of multiple subunits. The V1 sector contains subunits that hydrolyze ATP, whereas the V0 sector contains subunits that translocate protons across the membrane. Additional subunits in both sectors couple these activities. Here we have continued our examination of intermediate subunits primarily associated with the V1 but also implicated in interactions with the V0. Interactions between Vma7p (F) and Vma8p (D) and between Vma4p (E) and Vma10p (G) are described. Although Vma7p and Vma10p have been observed to interact with the V0 sector, our results indicate that these subunits behave primarily as canonical V1 sector subunits. We categorize these four subunits as "stalk" subunits to distinguish them from the known catalytic (A and B) and proton-translocating (c, c', and Vma16p) subunits and to highlight their intermediate nature. Furthermore, we show that the in vivo stability of Vma4p is dependent upon interaction with Vma10p. This may be important in the regulation of assembly, since these two subunits add to the V1 during later stages of V1 assembly. This is the first demonstration of interdependence between ATPase subunits for structural stability.

Reconstitution in Vitro of the V1 Complex from the Yeast Vacuolar Proton-translocating ATPase: ASSEMBLY RECAPITULATES MECHANISM

Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures. Assembly of soluble V1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits. Specifically, V1 can be assembled directly from complex III (subunits E and G) with complex IV (subunits A, B, D, and F) without prior disassembly of complex IV. The formation of complex III in vivo is also shown to be essential and could not be achieved in vitro. Assembly from simpler precursors is possible and is enhanced by added ATP. Assembly can be blocked by N-ethylmaleimide in a Vma1p (subunit A)-specific manner. From these data, we extend our previous model to consider an assembly pathway whose steps reflect the catalytic mechanism of the Boyer binding-change model.

Wild-type and Mutant Vacuolar Membranes Support pH-dependent Reassembly of the Yeast Vacuolar H+-ATPase in Vitro

Treatment of the yeast vacuolar proton-translocating ATPase (H+-ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane. Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30-70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5. Vacuolar membranes isolated from vma2Delta mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (Vo) complexes. The vma2Delta mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity. The results indicate that previously assembled Vo complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner.

Resolution of Subunit Interactions and Cytoplasmic Subcomplexes of the Yeast Vacuolar Proton-translocating ATPase

The vacuolar proton-translocating ATPase is the principal energization mechanism that enables the yeast vacuole to perform most of its physiological functions. We have undertaken an examination of subunit-subunit interactions and assembly states of this enzyme. Yeast two-hybrid data indicate that Vma1p and Vma2p interact with each other and that Vma4p interacts with itself. Three-hybrid data indicate that the Vma4p self-interaction is stabilized by both Vma1p and Vma2p. Native gel electrophoresis reveals numerous partial complexes not previously described. In addition to a large stable cytoplasmic complex seen in wild-type, Deltavma3 and Deltavma5 strains, we see partial complexes in the Deltavma4 and Deltavma7 strains. All larger complexes are lost in the Deltavma1, Deltavma2, and Deltavma8 strains. We designate the large complex seen in wild-type cells containing at least subunits Vma1p, Vma2p, Vma4p, Vma7p, and Vma8p as the definitive V1 complex.

Molecular genetics of the yeast vacuolar H(+)-ATPase.

The yeast vacuolar proton-translocating ATPase was discovered in 1981 as the first member of the V-ATPases, which are now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. Nine VMA genes that are indispensable for expression of vacuolar ATPase activity have been identified in the yeast Saccharomyces cerevisiae. VMA1, VMA2, VMA3, VMA5 and VMA6 were cloned and characterized on the basis of partial amino acid sequences determined with the purified subunits. Genetic and biochemical studies of the yeast Pet-cls mutants have demonstrated that they are related to vma defects. Based on this evidence, VMA11 (CLS9), VMA12 (CLS10) and VMA13 (CLS11) were isolated from a yeast genomic DNA library by complementation of the vma11, vma12 and vma13 mutations, respectively. This article summarizes currently available information on the VMA genes and the molecular biological functions of the VMA gene products.

Genetic and cell biological aspects of the yeast vacuolar H(+)-ATPase.

The yeast vacuolar proton-translocating ATPase is a member of the third class of H(+)-pumping ATPase. A family of this type of H(+)-ATPase is now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. Nine VMA genes that are indispensable for expression of the enzyme activity have been cloned and characterized in the yeast Saccharomyces cerevisiae. This review summarizes currently available information on the VMA genes and cell biological functions of the VMA gene products.

Subunit composition, biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase.

The yeast vacuole is acidified by a vacuolar proton-translocating ATPase (H(+)-ATPase) that closely resembles the vacuolar H(+)-ATPases of other fungi, animals, and plants. The yeast enzyme is purified as a complex of eight subunits, which include both integral and peripheral membrane proteins. The genes for seven of these subunits have been cloned, and mutant strains lacking each of the subunits (vma mutants) have been constructed. Disruption of any of the subunit genes appears to abolish the function of the vacuolar H(+)-ATPase, supporting the subunit composition derived from biochemical studies. Genetic studies of vacuolar acidification have also revealed an additional set of gene products that are required for vacuolar H(+)-ATPase activity, but may not be part of the final enzyme complex. The biosynthesis, assembly, and targeting of the enzyme is being elucidated by biochemical and cell biological studies of the vma mutants. Initial results suggest that the peripheral and integral membrane subunits may be independently assembled.

Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.

Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase.

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.

Role of Vacuolar Acidification in Protein Sorting and Zymogen Activation: a Genetic Analysis of the Yeast Vacuolar Proton-Translocating ATPase

Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.

Biochemical characterization of the yeast vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase was isolated by two different methods. A previously reported purification of the enzyme (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095) was repeated. This procedure consisted of isolation of vacuoles, solubilization with the zwitterionic detergent ZW3-14, and glycerol gradient centrifugation of the solubilized vacuoles. The fraction with the highest specific activity (11 mumol of ATP hydrolyzed mg-1 min-1) included eight polypeptides of apparent molecular masses of 100, 69, 60, 42, 36, 32, 27, and 17 kDa, suggesting that the enzyme may be more complex than the three-subunit composition proposed from the original purification. The 69-kDa polypeptide was recognized by antisera against the catalytic subunits of two other vacuolar ATPases and labeled with the ATP analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, indicating that it contains all or part of the catalytic site. A monoclonal antibody was prepared against this subunit. Under nondenaturing conditions, the antibody immunoprecipitated eight polypeptides, of the same molecular masses as those seen in the glycerol gradient fraction, from solubilized vacuolar vesicles. All eight of these polypeptides are therefore good candidates for being genuine subunits of the enzyme. The structure and function of the yeast vacuolar H+-ATPase were further characterized by examining the inhibition of ATPase activity by KNO3. In the presence of 5 mM MgATP, 100 mM KNO3 inhibited 71% of the ATPase activity of vacuolar vesicles, and the 69- and 60-kDa subunits (and possibly the 42-kDa subunit) were removed from the vacuolar membrane to a similar extent. At concentrations of less than 200 mM KNO3, the stripping of the ATPase subunits and the inhibition of ATPase activity were dependent on the presence of MgATP, suggesting that this is a conformation-specific disassembly of the enzyme. The yeast vacuolar H+-ATPase is a multisubunit enzyme, consisting of a combination of peripheral and integral membrane subunits. Its structure and subunit composition are very similar to other vacuolar ATPase, and it shares some characteristics with the F1F0-ATPases.