Transmembrane Topography of the 100-kDa a Subunit (Vph1p) of the Yeast Vacuolar Proton-translocating ATPase
1999; Elsevier BV; Volume: 274; Issue: 21 Linguagem: Inglês
10.1074/jbc.274.21.14655
ISSN1083-351X
AutoresXing-Hong Leng, Tsuyoshi Nishi, Michael Forgac,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe 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. 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. The vacuolar proton-translocating ATPases (or V-ATPases) 1The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; ACMA, 9-amino-6-chloro-2-methoxyacridine; MPB, 3-(N-maleimidylpropionyl) biocytin; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 2-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; ACMA, 9-amino-6-chloro-2-methoxyacridine; MPB, 3-(N-maleimidylpropionyl) biocytin; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 2-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. are multisubunit complexes found in a variety of intracellular compartments, such as clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles, chromaffin granules, synaptic vesicles, and the central vacuoles of yeast, Neurospora, and plants (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar, 2Forgac M. J. Bioenerg. Biomembr. 1992; 24: 341-350Crossref PubMed Scopus (50) Google Scholar, 3Bowman B.J. Vazquez-Laslop N. Bowman E.J. J. Bioenerg. Biomembr. 1992; 24: 361-370Crossref PubMed Scopus (54) Google Scholar, 4Kane P.M. Stevens T.H. J. Bioenerg. Biomembr. 1992; 24: 383-394Crossref PubMed Scopus (56) Google Scholar, 5Anraku Y. Umemoto N. Hirata R. Ohya Y. J. Bioenerg. Biomembr. 1992; 24: 395-406Crossref PubMed Scopus (63) Google Scholar, 6Sze H. Ward J.M. Lai S. J. Bioenerg. Biomembr. 1992; 24: 371-382Crossref PubMed Scopus (179) Google Scholar, 7Gluck S.L. J. Bioenerg. Biomembr. 1992; 24: 351-360Crossref PubMed Scopus (53) Google Scholar, 8Kibak H. Taiz L. Starke T. Bernasconi P. Gogarten J.P. J. Bioenerg. Biomembr. 1992; 24: 415-424Crossref PubMed Scopus (57) Google Scholar, 9Nelson N. J. Bioenerg. Biomembr. 1992; 24: 407-414Crossref PubMed Scopus (70) Google Scholar). Acidification of these intracellular compartments is in turn essential for a variety of cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of certain specialized cells, where they function in such processes as renal acidification (7Gluck S.L. J. Bioenerg. Biomembr. 1992; 24: 351-360Crossref PubMed Scopus (53) Google Scholar), bone resorption (10Chatterjee D. Chakraborty M. Leit M. Neff L. Jamsa-Kellokumpu S. Fuchs R. Baron R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6257-6261Crossref PubMed Scopus (132) Google Scholar), and pH homeostasis (11Swallow C.J. Grinstein S. Rotstein O.D. J. Biol. Chem. 1990; 265: 7645-7654Abstract Full Text PDF PubMed Google Scholar).The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two functional domains (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar). The V1 domain is a peripheral complex of molecular mass of 570 kDa composed of eight different subunits of molecular mass 70–14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular mass 100–17 kDa (subunits a, d, c, c′, and c“) that is responsible for proton translocation. This structure is similar to that of the ATP synthases (or F-ATPases) that function in ATP synthesis in mitochondria, chloroplasts and bacteria (12Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar, 13Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar, 14Cross R.L. Duncan T.M. J. Bioenerg. Biomembr. 1996; 28: 403-408Crossref PubMed Scopus (67) Google Scholar, 15Pedersen P.L. J. Bionerg. Biomembr. 1996; 28: 389-395Crossref PubMed Scopus (57) Google Scholar, 16Capaldi R.A. Aggeler R. Wilkens S. Gruber G. J. Bioenerg. Biomembr. 1996; 28: 397-401Crossref PubMed Scopus (61) Google Scholar, 17Futai M. Omote H. J. Bioenerg. Biomembr. 1996; 28: 409-414Crossref PubMed Scopus (31) Google Scholar), and sequence homology between these classes of ATPase has been demonstrated for both the nucleotide binding subunits (A, B, α, and β) (18Zimniak L. Dittrich P. Gogarten J.P. Kibak H. Taiz L. J. Biol. Chem. 1988; 263: 9102-9112Abstract Full Text PDF PubMed Google Scholar, 19Bowman E.J. Tenney K. Bowman B. J. Biol. Chem. 1988; 263: 13994-14001Abstract Full Text PDF PubMed Google Scholar) and the proteolipid subunits (subunits c, c′, and c”) (20Mandel M. Moriyama Y. Hulmes J.D. Pan Y.C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5521-5524Crossref PubMed Scopus (238) Google Scholar, 21Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). No sequence homology has been identified for any of the remaining subunits.The 100-kDa subunit of the V-ATPase is an integral membrane protein possessing an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain containing multiple putative membrane spanning segments (22Perin M.S. Fried V.A. Stone D.K. Xie X.S. Sudhof T.C. J. Biol. Chem. 1991; 266: 3877-3881Abstract Full Text PDF PubMed Google Scholar, 23Manolson M.F. Proteau D. Preston R.A. Stenbit A. Roberts B.T. Hoyt M. Preuss D. Mulholland J. Botstein D. Jones E.W. J. Biol. Chem. 1992; 267: 14294-14303Abstract Full Text PDF PubMed Google Scholar, 24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar). In yeast, the 100-kDa subunit is encoded by two genes, VPH1 and STV1 (23Manolson M.F. Proteau D. Preston R.A. Stenbit A. Roberts B.T. Hoyt M. Preuss D. Mulholland J. Botstein D. Jones E.W. J. Biol. Chem. 1992; 267: 14294-14303Abstract Full Text PDF PubMed Google Scholar, 24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar). Vph1p is targeted to the central vacuole, whereas Stv1p is normally targeted to some other intracellular membrane, possibly Golgi or endosomes (24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar).We have previously demonstrated using a combination of site-directed and random mutagenesis that Vph1p contains several buried charged residues, mutation of which significantly alters proton transport activity of the V-ATPase complex (25Leng X.H. Manolson M. Liu Q. Forgac M. J. Biol. Chem. 1996; 271: 22487-22493Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 26Leng X.H. Manolson M. Forgac M. J. Biol. Chem. 1998; 273: 6717-6723Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). These results have led us to suggest that the 100-kDa subunit is the V-ATPase homolog to the a subunit of the F-ATPases. Mutational studies have identified several critical buried charged residues in the last two transmembrane segments of the F-ATPase a subunit that are important for proton translocation (27Cain B.D. Simoni R.D. J. Biol. Chem. 1986; 261: 10043-10050Abstract Full Text PDF PubMed Google Scholar, 28Cain B.D. Simoni R.D. J. Biol. Chem. 1988; 263: 6606-6612Abstract Full Text PDF PubMed Google Scholar, 29Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Abstract Full Text PDF PubMed Google Scholar, 30Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar), although more recent studies have suggested that some of these residues can be replaced without complete loss of function (31Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1997; 272: 32635-32641Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar).Although considerable information has been obtained concerning the membrane topography of the F-ATPase a subunit (32Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1998; 273: 16241-16247Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 33Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), essentially no information has yet been reported concerning the folding of the V-ATPase a subunit. In the present study, we have employed a combination of cysteine-scanning mutagenesis and chemical modification together with introduction of factor Xa cleavage sites in putative loops to investigate the topographical arrangement of the V-ATPase a subunit.DISCUSSIONWe have used cysteine-scanning mutagenesis together with chemical modification by membrane permeable and impermeable reagents, as well as introduction of protease cleavage sites, to analyze the membrane topography of the 100-kDa a subunit of the yeast V-ATPase. The cysteine-scanning method depends upon the successful construction of a functionally active Cys-less form of the protein together with mutants containing unique cysteine residues that do not significantly perturb activity. In the case of the a subunit, this required the simultaneous replacement of seven endogenous cysteine residues. In addition, because the a subunit is part of a multisubunit complex, the mutations introduced must not alter the interactions between the a subunit and the remaining V-ATPase subunits. We observed that both the Cys-less form of the a subunit and all but one of the single cysteine-containing mutants give rise to complexes showing near wild type levels of activity, suggesting that these changes do not significantly alter the structure or function of the a subunit. In the case of G763C, a loss of approximately 50% of proton transport activity was observed.The labeling strategy employed depends upon the membrane permeability of MPB and the membrane impermeability of AMS. This allows MPB to react with cysteine residues on both sides of the membrane, whereas AMS reacts only with cysteine residues exposed on the surface of the membrane vesicles. In the case of yeast vacuolar membrane vesicles used in the present study, the exposed surface is the cytoplasmic side of the membrane. The orientation and sealed state of the vesicles employed is supported by the similar ratio of proton transport to ATP hydrolysis that we have observed when compared with other sealed, well oriented vesicles, including intact yeast vacuoles (46Liu Q. Kane P.M. Newman P.R. Forgac M. J. Biol. Chem. 1996; 271: 2018-2022Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and purified clathrin-coated vesicles (47Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar). This strategy has been successfully used to study the membrane topography of several other integral membrane proteins, including in particular subunit a of the E. coliF-ATPase (32Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1998; 273: 16241-16247Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 33Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These latter studies have led to the current five membrane spanning model for the F-ATPase a subunit.In addition to the cysteine modification approach, we have also introduced factor Xa cleavage sites into three putative loop regions of the protein. Although the mutant bearing a factor Xa site at position 606 showed only approximately 50% of wild type levels of activity, the other two mutants showed nearly normal proton transport, suggesting minimal perturbation of structure.The results obtained in the current study have led to the model for the folding of the V-ATPase a subunit shown in Fig. 4. In addition, a hydropathy plot for Vph1p together with the location of each of the putative membrane spanning segments is shown in Fig. 5. The protein is proposed to span the bilayer nine times, with the amino terminus on the cytoplasmic side of the membrane and the carboxyl terminus on the lumenal side. All four of the cysteine residues introduced into the amino-terminal soluble domain showed a labeling pattern consistent with a cytoplasmic orientation whereas S840C at the carboxyl terminus showed labeling characteristic of a lumenal orientation. This suggests that the amino and carboxyl termini are on opposite sides of the membrane, requiring that the protein span the bilayer an odd number of times.Figure 5Hydropathy plot of Vph1p and location of putative transmembrane segments. The amino acid sequence of Vph1p (24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar) was analyzed using the method of Kyte and Doolittle (49Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17007) Google Scholar), and the resultant hydropathy plot shown. The location of the nine putative transmembrane segments are also shown and labeledI--IX. There are four consensusN-linked glycosylation sites (Asn-X-Ser/Thr) in Vph1p at Asn-113, Asn-280, Asn-324, and Asn-374. Because all four sites are located in the amino-terminal soluble domain, which our labeling data clearly shows is cytoplasmic, it is unlikely that any of these four sites are glycosylated in vivo. In fact, it has not clearly been demonstrated that Vph1p is glycosylated in yeast. If Vph1p is glycosylated, it is possible that carbohydrate is attached atO-linked sites, for which there is not a clear consensus sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In our original folding model for the a subunit (25Leng X.H. Manolson M. Liu Q. Forgac M. J. Biol. Chem. 1996; 271: 22487-22493Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), we had postulated that the amino-terminal soluble domain was exposed on the lumenal side of the membrane based upon the increased labeling observed for the bovine a subunit using membrane impermeant reagents when the membrane was disrupted with detergents (47Arai H. Terres G. Pink S. Forgac M. J. Biol. Chem. 1988; 263: 8796-8802Abstract Full Text PDF PubMed Google Scholar). This result may be explained by a change in conformation of the protein in the presence of detergent such that previously shielded sites on the cytoplasmic side of the membrane become exposed. In addition, because the a subunit is not synthesized with an amino-terminal leader sequence, a cytoplasmic orientation for the amino terminus is more consistent with what is known concerning the requirements for translocation of hydrophilic protein segments across the endoplasmic reticulum membrane during biosynthesis.A lumenal orientation of the carboxyl terminus is also at odds with our previously proposed model, in which we suggested a cytoplasmic orientation for the carboxyl terminus (26Leng X.H. Manolson M. Forgac M. J. Biol. Chem. 1998; 273: 6717-6723Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This was based on the identification by random mutagenesis of a cluster of five mutations between residues Leu-800 and Gly-814 that disrupted attachment of the V1 and V0 domains, suggesting that this region may be important in assembly of the V-ATPase complex (26Leng X.H. Manolson M. Forgac M. J. Biol. Chem. 1998; 273: 6717-6723Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is nevertheless possible that disruption of assembly in these mutants results from conformational changes in the structure of the 100-kDa subunit or the V0 domain that prevent attachment of V1 to V0. Interestingly, our current model places nearly all of the residues that have been observed to disrupt assembly, including Asp-425, Lys-538, and Arg-735, as well as the five mutations between residues 800 and 814 on the lumenal side of the membrane. It is possible that these residues may form a lumenal domain that senses the intravesicular pH and conveys this information through conformational changes in the 100-kDa subunit to the cytoplasmic domain of the complex.With respect to the loop regions, four of the introduced cysteine residues (at positions 561, 564, 761, and 763) are not labeled by MPB, suggesting that they are shielded from reaction by interaction with other V0 subunits or other regions of the 100-kDa subunit itself. It is also possible that one or more of these cysteine residues may be shielded from reaction with MPB by interaction with the lipid bilayer. Of the remaining introduced cysteine residues, three appear to have a cytoplasmic orientation (N447C, N450C, and S703C) whereas G602C appears to be lumenal. The cytoplasmic orientation of residues 447 and 450 requires that the hydrophobic region between residues 405 and 443 span the bilayer twice rather than once (as originally postulated (25Leng X.H. Manolson M. Liu Q. Forgac M. J. Biol. Chem. 1996; 271: 22487-22493Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar)), placing Asp-425 on the lumenal side of the membrane. A lumenal orientation for Gly-602 and a cytoplasmic orientation for Ser-703 is consistent with a single membrane span between these sites. That Ser-703 is cytoplasmic is also consistent with the observation that the bovine V-ATPase a subunit can be cleaved by low concentrations of trypsin at a site immediately upstream of this residue in intact coated vesicles (48Adachi I. Arai H. Pimental R. Forgac M. J. Biol. Chem. 1990; 265: 960-966Abstract Full Text PDF PubMed Google Scholar). Thus, the large soluble loop between residues 653 and 727 is likely to be oriented toward the cytoplasm.Of the three factor Xa sites introduced into the 100-kDa subunit, protease cleavage was observed only for the tandem sites introduced at position 560. The fact that this cleavage happened in intact vacuolar membrane vesicles as well as in the presence of detergent indicates that this site is exposed on the cytoplasmic side of the membrane. For the sites introduced at positions 499 and 606, no cleavage by factor Xa was observed in either the presence or absence of detergent, again suggesting that these sites may be shielded through interaction with other regions of the protein. Nevertheless, the observation that all three of these mutants show significant proton transport activity suggests that all three sites are located in loop regions between transmembrane segments in the a subunit. This conclusion comes from results obtained with lac permease, where it has been observed that factor Xa sites introduced into loops between transmembrane segments generally do not affect activity, whereas sites introduced within transmembrane segments invariably lead to loss of both correct folding and transport activity. 3R. Kaback, personal communication. It is important to emphasize that the model shown in Fig. 4 is only a working model for the topography of the V-ATPase a subunit based upon the first available data presented here concerning the disposition of this polypeptide with respect to the membrane. The labeling and proteolysis approaches taken in the current study, like all other methods of analyzing the topography of membrane proteins, are subject to several possible sources of error. For instance, it is possible that local differences in the environment of individual cysteine residues, in addition to their disposition with respect to the membrane, may influence their relative reactivity toward the two sulfhydryl reagents employed. We have shown that permeabilization of the membranes with detergent allows the lumenally oriented cysteine residues to be effectively blocked by AMS. Nevertheless, it will be important that the proposed model be further tested, both by introduction of additional labeling and cleavage sites and by employing other approaches to determine topography, such as epitope tagging and preparation of site-specific antibodies that recognize the native protein. The current study, however, represents a first step in determining the folding pattern of this important V-ATPase subunit. The vacuolar proton-translocating ATPases (or V-ATPases) 1The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; ACMA, 9-amino-6-chloro-2-methoxyacridine; MPB, 3-(N-maleimidylpropionyl) biocytin; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 2-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; ACMA, 9-amino-6-chloro-2-methoxyacridine; MPB, 3-(N-maleimidylpropionyl) biocytin; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 2-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. are multisubunit complexes found in a variety of intracellular compartments, such as clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles, chromaffin granules, synaptic vesicles, and the central vacuoles of yeast, Neurospora, and plants (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar, 2Forgac M. J. Bioenerg. Biomembr. 1992; 24: 341-350Crossref PubMed Scopus (50) Google Scholar, 3Bowman B.J. Vazquez-Laslop N. Bowman E.J. J. Bioenerg. Biomembr. 1992; 24: 361-370Crossref PubMed Scopus (54) Google Scholar, 4Kane P.M. Stevens T.H. J. Bioenerg. Biomembr. 1992; 24: 383-394Crossref PubMed Scopus (56) Google Scholar, 5Anraku Y. Umemoto N. Hirata R. Ohya Y. J. Bioenerg. Biomembr. 1992; 24: 395-406Crossref PubMed Scopus (63) Google Scholar, 6Sze H. Ward J.M. Lai S. J. Bioenerg. Biomembr. 1992; 24: 371-382Crossref PubMed Scopus (179) Google Scholar, 7Gluck S.L. J. Bioenerg. Biomembr. 1992; 24: 351-360Crossref PubMed Scopus (53) Google Scholar, 8Kibak H. Taiz L. Starke T. Bernasconi P. Gogarten J.P. J. Bioenerg. Biomembr. 1992; 24: 415-424Crossref PubMed Scopus (57) Google Scholar, 9Nelson N. J. Bioenerg. Biomembr. 1992; 24: 407-414Crossref PubMed Scopus (70) Google Scholar). Acidification of these intracellular compartments is in turn essential for a variety of cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of certain specialized cells, where they function in such processes as renal acidification (7Gluck S.L. J. Bioenerg. Biomembr. 1992; 24: 351-360Crossref PubMed Scopus (53) Google Scholar), bone resorption (10Chatterjee D. Chakraborty M. Leit M. Neff L. Jamsa-Kellokumpu S. Fuchs R. Baron R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6257-6261Crossref PubMed Scopus (132) Google Scholar), and pH homeostasis (11Swallow C.J. Grinstein S. Rotstein O.D. J. Biol. Chem. 1990; 265: 7645-7654Abstract Full Text PDF PubMed Google Scholar). The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two functional domains (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar). The V1 domain is a peripheral complex of molecular mass of 570 kDa composed of eight different subunits of molecular mass 70–14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular mass 100–17 kDa (subunits a, d, c, c′, and c“) that is responsible for proton translocation. This structure is similar to that of the ATP synthases (or F-ATPases) that function in ATP synthesis in mitochondria, chloroplasts and bacteria (12Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar, 13Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar, 14Cross R.L. Duncan T.M. J. Bioenerg. Biomembr. 1996; 28: 403-408Crossref PubMed Scopus (67) Google Scholar, 15Pedersen P.L. J. Bionerg. Biomembr. 1996; 28: 389-395Crossref PubMed Scopus (57) Google Scholar, 16Capaldi R.A. Aggeler R. Wilkens S. Gruber G. J. Bioenerg. Biomembr. 1996; 28: 397-401Crossref PubMed Scopus (61) Google Scholar, 17Futai M. Omote H. J. Bioenerg. Biomembr. 1996; 28: 409-414Crossref PubMed Scopus (31) Google Scholar), and sequence homology between these classes of ATPase has been demonstrated for both the nucleotide binding subunits (A, B, α, and β) (18Zimniak L. Dittrich P. Gogarten J.P. Kibak H. Taiz L. J. Biol. Chem. 1988; 263: 9102-9112Abstract Full Text PDF PubMed Google Scholar, 19Bowman E.J. Tenney K. Bowman B. J. Biol. Chem. 1988; 263: 13994-14001Abstract Full Text PDF PubMed Google Scholar) and the proteolipid subunits (subunits c, c′, and c”) (20Mandel M. Moriyama Y. Hulmes J.D. Pan Y.C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5521-5524Crossref PubMed Scopus (238) Google Scholar, 21Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). No sequence homology has been identified for any of the remaining subunits. The 100-kDa subunit of the V-ATPase is an integral membrane protein possessing an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain containing multiple putative membrane spanning segments (22Perin M.S. Fried V.A. Stone D.K. Xie X.S. Sudhof T.C. J. Biol. Chem. 1991; 266: 3877-3881Abstract Full Text PDF PubMed Google Scholar, 23Manolson M.F. Proteau D. Preston R.A. Stenbit A. Roberts B.T. Hoyt M. Preuss D. Mulholland J. Botstein D. Jones E.W. J. Biol. Chem. 1992; 267: 14294-14303Abstract Full Text PDF PubMed Google Scholar, 24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar). In yeast, the 100-kDa subunit is encoded by two genes, VPH1 and STV1 (23Manolson M.F. Proteau D. Preston R.A. Stenbit A. Roberts B.T. Hoyt M. Preuss D. Mulholland J. Botstein D. Jones E.W. J. Biol. Chem. 1992; 267: 14294-14303Abstract Full Text PDF PubMed Google Scholar, 24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar). Vph1p is targeted to the central vacuole, whereas Stv1p is normally targeted to some other intracellular membrane, possibly Golgi or endosomes (24Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar). We have previously demonstrated using a combination of site-directed and random mutagenesis that Vph1p contains several buried charged residues, mutation of which significantly alters pr
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