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W1999036031 abstract "The effect of vacuolar H+-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H+-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed. The effect of vacuolar H+-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H+-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed. vacuolar H+-ATPase 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid kilobase pair(s) endoplasmic reticulum wild type Proton pumps play a major role in providing energy for several secondary uptake processes as well as maintaining the pH homeostasis required for the living cell and subcellular compartments (1Nelson N. Harvey W.R. Phys. Rev. 1999; 79: 361-385Crossref PubMed Scopus (367) Google Scholar). In the yeast Saccharomyces cerevisiae, there are two mechanistically distinct ATP-dependent proton pumps that play a pivotal role in these processes. One is Pma1p, which functions in the plasma membrane, and the second is the V-ATPase,1 which functions in the vacuolar system (2Stevens T.H. Forgac M. Annu. Rev. Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar, 3Catty P. de Kerchove d'Exaerde A. Goffeau A. FEBS Lett. 1997; 409: 325-332Crossref PubMed Scopus (110) Google Scholar). Pma1p is an essential enzyme that provides the protonmotive force for the yeast plasma membrane and plays a major role in the pH homeostasis of the cell (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). The enzyme is capable of generating very high membrane potentials, and in the presence of permeant anions, pH gradients of more than 3 units are observed (5Slayman C.L. J. Bioenerg. Biomembr. 1987; 19: 1-20PubMed Google Scholar). To maintain its proper activity as well as to prevent its deleterious activity in different aspects of the secretory pathway, its transport to the cell surface must be strictly controlled. The amount of Pma1p in the plasma membrane is also under strict control, and usually the enzyme is present at relatively constant amounts of 25–50% of the membrane proteins (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). Because of its importance for the viability of the cell, Pma1p was subjected to extensive studies aimed at understanding its mechanism of action as well as its potential as a drug target for antifungal agents (6Seto-Young D. Monk B. Mason A.B. Perlin D.S. Biochim. Biophys. Acta. 1997; 1326: 249-256Crossref PubMed Scopus (52) Google Scholar). For these reasons, the genePMA1 was subjected to mutagenesis and numerous mutants that influenced its catalytic activity and/or targeting to the plasma membrane have been generated (7Petrov V.V. Slayman C.W. J. Biol. Chem. 1995; 270: 28535-28540Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8Rao R. Slayman C.W. J. Biol. Chem. 1993; 268: 6708-6713Abstract Full Text PDF PubMed Google Scholar, 9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 10Ambesi A. Miranda M. Petrov V.V. Slayman C.W. J. Exp. Biol. 2000; 203: 155-160PubMed Google Scholar). Some of these mutants exhibited reduced amounts of Pma1p in the plasma membrane and resulted in growth sensitivity at low pH (11Perlin D.S. Harris S.L. Monk B.C. Seto-Young D. Na S. Anand S. Haber J.E. Acta Physiol. Scand. 1992; 607: 183-192Google Scholar, 12McCusker J.H. Perlin D.S. Haber J.E. Mol. Cell. Biol. 1987; 7: 4082-4088Crossref PubMed Scopus (138) Google Scholar).While Pma1p is vital, and null mutations in this protein are lethal, null mutations even in one of the numerous V-ATPase subunits, while lethal in other eukaryotes, are conditionally lethal in yeast. Their vitality can be sustained when yeast mutants are grown at low pH (13Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar, 14Nelson H. Mandiyan S. Nelson N. J. Biol. Chem. 1994; 269: 24150-24155Abstract Full Text PDF PubMed Google Scholar, 15Nelson H. Mandiyan S. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 497-501Crossref PubMed Scopus (77) Google Scholar). It was proposed that yeast V-ATPase null mutants could grow at low pH because they are able to energize their vacuolar system by fluid phase endocytosis, which provides an acidic medium for the lumen. Indeed, inactivation of the genes involved in endocytosis on the background of a V-ATPase null mutation caused lethality (16Munn A.L. Riezman H. J. Cell Biol. 1994; 127: 373-386Crossref PubMed Scopus (229) Google Scholar). Due to its location in the organelles of the secretory pathway, the V-ATPase plays a role in protein targeting (17Forgac M. FEBS Lett. 1998; 440: 258-263Crossref PubMed Scopus (118) Google Scholar). Inhibition of the V-ATPase with the drug bafilomycin A1, as well as V-ATPase null mutations, resulted in the accumulation and missorting of precursor forms of various vacuolar proteins (18Banta L.M. Robinson J.S. Klionsky D.J. Emr S.D. J. Cell Biol. 1988; 107: 1369-1383Crossref PubMed Scopus (299) Google Scholar, 19Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (145) Google Scholar, 20Umemoto N. Yoshihisa T. Hirata R. Anraku Y. J. Biol. Chem. 1990; 265: 18447-18453Abstract Full Text PDF PubMed Google Scholar, 21Klionsky D.J. Nelson H. Nelson N. J. Biol. Chem. 1992; 267: 3416-3422Abstract Full Text PDF PubMed Google Scholar, 22Yaver D.S. Nelson H. Nelson N. Klionsky D.J. J. Biol. Chem. 1993; 268: 10564-10572Abstract Full Text PDF PubMed Google Scholar). In addition, it was shown that, under those conditions, precursor forms of vacuolar hydrolases were accumulated within the Golgi complex and/or post-Golgi compartments (22Yaver D.S. Nelson H. Nelson N. Klionsky D.J. J. Biol. Chem. 1993; 268: 10564-10572Abstract Full Text PDF PubMed Google Scholar) and that proteins having luminally oriented targeting signals were directly dependent on compartment acidification for efficient vacuolar delivery (23Morano K.A Klionsky D.J. J. Cell Sci. 1994; 107: 2813-2824Crossref PubMed Google Scholar).Studies with different vacuolar proteins revealed involvement of at least two distinct targeting pathways to the vacuole (24Wendland B. Emr S.D. Riezman H. Curr. Opin. Cell Biol. 1998; 10: 513-522Crossref PubMed Scopus (150) Google Scholar). One goes through the endosomes, and the other delivers the proteins directly to the vacuole from the post-Golgi vesicles. Similarly, multiple delivery pathways of plasma membrane proteins may exist (25de Kerchove d'Exaerde A. Supply P. Goffeau A. Yeast. 1996; 12: 907-916Crossref PubMed Scopus (30) Google Scholar). Studies of Pma1p sorting revealed involvement of several proteins needed specifically for its transport to the plasma membrane (9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 26Luo W.J. Chang A. J. Cell Biol. 1997; 138: 731-746Crossref PubMed Scopus (91) Google Scholar, 27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). In this work, we report on such a differential effect of V-ATPase null mutations on the sorting of Pma1p to the plasma membrane. We show that while the targeting of Pma1p is highly affected by the V-ATPase null mutations, these mutations have very little effect on the targeting of Gas1p, Sec61p, or Sed5p into their respective compartments.Until now, every V-ATPase mutant studied that abolished the activity of the enzyme shared more then one feature with the other V-ATPase mutants' phenotypes. Since medium pH growth requirements are common to a variety of different mutations, in order to characterize a new V-ATPase-defective mutant, we have to demonstrate several features that coexist with and are similar to a known subunit of the V-ATPase-deleted mutant. The sorting profile of Pma1p in the V-ATPase mutants can be added to this assortment of features.Kex2p and Gef1p, two membrane proteins residing in the post-Golgi vesicles, were indicated as influencing V-ATPase activity. Their null mutants were created in two different yeast strains, and both had intact V-ATPase activity and regular quinacrine accumulation in the vacuole as well as wild-type profile of Pma1p distribution. The conclusions from these observations are that neither Kex2p nor Gef1p has a general effect upon the V-ATPase activity; nor do they affect the sorting path of Pma1p in the secretory pathway in the way that the V-ATPase null mutant does.DISCUSSIONPma1p is an essential enzyme that provides the protonmotive force for the yeast plasma membrane and plays a major role in the pH homeostasis of the cell. The amount of Pma1p in the plasma membrane is also under strict control, and the enzyme is present usually in a relatively constant amount (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). The V-ATPase complements the function of Pma1p in organelles and membranes of the vacuolar system (52Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar). In addition to its function as the main energizing enzyme in the vacuolar system, the V-ATPase was found to be involved in the sorting processes of proteins to the vacuolar compartment in yeast (53Nelson N. Klionsky D.J. Experientia. 1996; 52: 1101-1110Crossref PubMed Scopus (33) Google Scholar). It has long been known that acidification of a lysosomal compartment is required for proper sorting of soluble lysosomal proteases in mammalian cells, utilizing the mannose 6-phosphate receptor pathway (54Mellman I. J. Exp. Biol. 1992; 172: 39-45Crossref PubMed Google Scholar, 55Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1331) Google Scholar). The pre-Golgi and cis-Golgi membranes were also shown to contain a V-ATPase activity. There it regulates retrograde membrane traffic at the ER-Golgi pathway (56Palokangas H. Ying M. Vaananen K. Saraste J. Mol. Biol. Cell. 1998; 9: 3561-3578Crossref PubMed Scopus (86) Google Scholar). A compartment acidification requirement was demonstrated in the targeting of GPP130 (Golgiphosphoprotein of 130 kDa) to the early Golgi (57Linstedt A.D. Mehta A. Suhan J. Reggio H. Hauri H.P. Mol. Biol. Cell. 1997; 8: 1073-1087Crossref PubMed Scopus (99) Google Scholar). It would be expected that the other secretory sites would also be sensitive to the pH in their respective compartments.In this study, we examined the influence of V-ATPase null mutations in yeast on the processes of targeting to the plasma membrane by following their effect on the Pma1p distribution. Numerous studies investigated the influence of deletion or overexpression of proteins, known to reside in the secretory pathway, on Pma1p sorting and targeting to the plasma membrane in yeast. Among them, Ast1p, Ast2p, Vps8p, and Vps36p were investigated and found to influence the Pma1p distribution (9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 58Luo W.J. Chang A. Mol. Biol. Cell. 2000; 11: 579-592Crossref PubMed Scopus (63) Google Scholar). By comparison with the Gas1p, another plasma membrane resident, several proteins with a differential effect on Pma1p were discovered. Among them is Lst1p, which was suggested to take part in the transport of Pma1p from ER to the Golgi compartment (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). Another was identified as Eps1p (ER-retained PMA1suppressor), which functions in ER quality control (45Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). The mutant of Eps1p suppresses the D378N phenotype by allowing both the mutant and wild-type Pma1p molecules to travel to the plasma membrane. Its null mutant does not affect the sorting of native Pma1p at all, whereas it causes a marked accumulation of the 105-kDa ER form of Gas1p in the ER.We found that V-ATPase null mutants have a specific effect on Pma1p as well. To determine its distribution on the sucrose gradient, we used Sec61p as a marker of the ER, Sed5p as a marker for the Golgi compartment, and Gas1p as the other plasma membrane marker. There was no change in the distribution of Sec61p and Sed5p in the mutant in comparison with the wild-type strain; nor was the amount of Gas1p in the plasma membrane reduced, although its 105-kDa intermediate was elevated in the ER in comparison with the wild-type strain. However, Pma1p amounts in the plasma membrane in the ATPase-depleted mutants were markedly reduced, and a large amount of the protein was accumulated in the ER in a nonactive form.The fact that, in V-ATPase null mutants, the distribution of Gas1p differs from Pma1p suggests that lack of acidification of the various compartments differentially changes the protein composition of their membranes but not the characteristics of the membranes in general. The decline in Pma1p in plasma membrane may be a unique feature of this protein, whereas the slowdown of movement from ER to Golgi, as demonstrated by Gas1p in the mutant yeast, might be a general feature of the mutant.The question remains as to the site of the crucial secretory pathway compartment, whose acidification by V-ATPase is vital. The fact that the site of Pma1p accumulation in the mutant is in the ER suggests that the passage from ER to Golgi in the V-ATPase mutants is impaired. This hypothesis is supported by several previous results. It was shown in mammalian cells that retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the V-ATPase-specific inhibitor Bafilomycin A1 (56Palokangas H. Ying M. Vaananen K. Saraste J. Mol. Biol. Cell. 1998; 9: 3561-3578Crossref PubMed Scopus (86) Google Scholar). In yeast, the deletion of the LST1 gene that encodes for a peripheral ER membrane protein results in a profile of Pma1p distribution on sucrose gradient similar to the one of V-ATPase null mutants (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). It was suggested that Lst1p takes part in efficient packaging of Pma1p into vesicles derived from the ER. Moreover, it was shown that the plasma membrane H+-ATPase undergoes pH-dependent conformational changes (59Blanpain J.P. Ronjat M. Supply P. Dufour J.P. Goffeau A. Dupont Y. J. Biol. Chem. 1992; 267: 3735-3740Abstract Full Text PDF PubMed Google Scholar). If we assume that the V-ATPase functions incis-Golgi, then the impaired sorting of Pma1p in the V-ATPase null mutant could be explained by damage to the retrograde transport of factors such as Lst1p or conformational changes in Pma1p, imposed by the improper pH conditions.The defect that V-ATPase null mutants are mostly checked for is their inability to grow on a buffered pH 7.5 medium. However, these mutants are also defective in growth on a low pH medium (13Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar). The inability to grow on very acidic medium was demonstrated for a wide range of Pma1p mutants as well and was explained by a low activity of the mutant, stemming from either the mutation or competition of the mutant on the targeting to the membrane with the intact protein (12McCusker J.H. Perlin D.S. Haber J.E. Mol. Cell. Biol. 1987; 7: 4082-4088Crossref PubMed Scopus (138) Google Scholar, 60Wang G. Tamas M.J. Hall M.J. Pascual-Ahuir A. Perlin D.S. J. Biol. Chem. 1996; 271: 25438-25445Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Since we find that in V-ATPase null mutants the amount of Pma1p in the plasma membrane is markedly reduced, it is tempting to suggest that the reduced growth rate at low pH is caused by reduced levels of Pma1p in the plasma membrane of those mutants.In this study, we show that the altered distribution of Pma1p in V-ATPase null mutant can serve as another characteristic feature of it. Kex2p and Gef1p were both suggested to be involved in the proper function of the acidification by the V-ATPase (48Oluwatosin Y.E. Kane P.M. Mol. Cell. Biol. 1998; 18: 1534-1543Crossref PubMed Scopus (30) Google Scholar, 49Gaxiola R.A. Yuan D.S. Klausner R.D. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4046-4050Crossref PubMed Scopus (148) Google Scholar). In the case of the ΔKEX2 null mutant, there has been a discrepancy in the original paper, where the ATPase and proton pumping activities in isolated vacuoles of this mutant were undamaged, whereas the vacuoles failed to accumulate quinacrine. To exclude the possibility that in our strain (W303), uniquely, the deletion of Kex2p did not affect the quinacrine accumulation in the vacuoles, we disrupted this gene in two additional strains and in them observed as well that the ΔKEX2 mutant was fully active in quinacrine accumulation into their vacuoles. In addition, when we checked the distribution of the Pma1p in theΔKEX2 and ΔGEF1mutants, it was similar to the wild type and not to the V-ATPase mutant. By metal supplementation in the growth medium, we showed that the addition of 5 μm Cu2+ rescued theΔKEX2 and ΔGEF1 mutants and enabled their growth on pH 7.5 medium, whereas it did not have any effect upon the V-ATPase mutant. Therefore, a direct interaction between Kex2p or Gef1p and V-ATPase can be excluded. However, their activity may be involved in some cellular functions, such as anion transport or metal ion homeostasis, in specific organelles of the secretory pathway that also affect the growth at high pH levels in the medium. Proton pumps play a major role in providing energy for several secondary uptake processes as well as maintaining the pH homeostasis required for the living cell and subcellular compartments (1Nelson N. Harvey W.R. Phys. Rev. 1999; 79: 361-385Crossref PubMed Scopus (367) Google Scholar). In the yeast Saccharomyces cerevisiae, there are two mechanistically distinct ATP-dependent proton pumps that play a pivotal role in these processes. One is Pma1p, which functions in the plasma membrane, and the second is the V-ATPase,1 which functions in the vacuolar system (2Stevens T.H. Forgac M. Annu. Rev. Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (520) Google Scholar, 3Catty P. de Kerchove d'Exaerde A. Goffeau A. FEBS Lett. 1997; 409: 325-332Crossref PubMed Scopus (110) Google Scholar). Pma1p is an essential enzyme that provides the protonmotive force for the yeast plasma membrane and plays a major role in the pH homeostasis of the cell (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). The enzyme is capable of generating very high membrane potentials, and in the presence of permeant anions, pH gradients of more than 3 units are observed (5Slayman C.L. J. Bioenerg. Biomembr. 1987; 19: 1-20PubMed Google Scholar). To maintain its proper activity as well as to prevent its deleterious activity in different aspects of the secretory pathway, its transport to the cell surface must be strictly controlled. The amount of Pma1p in the plasma membrane is also under strict control, and usually the enzyme is present at relatively constant amounts of 25–50% of the membrane proteins (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). Because of its importance for the viability of the cell, Pma1p was subjected to extensive studies aimed at understanding its mechanism of action as well as its potential as a drug target for antifungal agents (6Seto-Young D. Monk B. Mason A.B. Perlin D.S. Biochim. Biophys. Acta. 1997; 1326: 249-256Crossref PubMed Scopus (52) Google Scholar). For these reasons, the genePMA1 was subjected to mutagenesis and numerous mutants that influenced its catalytic activity and/or targeting to the plasma membrane have been generated (7Petrov V.V. Slayman C.W. J. Biol. Chem. 1995; 270: 28535-28540Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8Rao R. Slayman C.W. J. Biol. Chem. 1993; 268: 6708-6713Abstract Full Text PDF PubMed Google Scholar, 9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 10Ambesi A. Miranda M. Petrov V.V. Slayman C.W. J. Exp. Biol. 2000; 203: 155-160PubMed Google Scholar). Some of these mutants exhibited reduced amounts of Pma1p in the plasma membrane and resulted in growth sensitivity at low pH (11Perlin D.S. Harris S.L. Monk B.C. Seto-Young D. Na S. Anand S. Haber J.E. Acta Physiol. Scand. 1992; 607: 183-192Google Scholar, 12McCusker J.H. Perlin D.S. Haber J.E. Mol. Cell. Biol. 1987; 7: 4082-4088Crossref PubMed Scopus (138) Google Scholar). While Pma1p is vital, and null mutations in this protein are lethal, null mutations even in one of the numerous V-ATPase subunits, while lethal in other eukaryotes, are conditionally lethal in yeast. Their vitality can be sustained when yeast mutants are grown at low pH (13Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar, 14Nelson H. Mandiyan S. Nelson N. J. Biol. Chem. 1994; 269: 24150-24155Abstract Full Text PDF PubMed Google Scholar, 15Nelson H. Mandiyan S. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 497-501Crossref PubMed Scopus (77) Google Scholar). It was proposed that yeast V-ATPase null mutants could grow at low pH because they are able to energize their vacuolar system by fluid phase endocytosis, which provides an acidic medium for the lumen. Indeed, inactivation of the genes involved in endocytosis on the background of a V-ATPase null mutation caused lethality (16Munn A.L. Riezman H. J. Cell Biol. 1994; 127: 373-386Crossref PubMed Scopus (229) Google Scholar). Due to its location in the organelles of the secretory pathway, the V-ATPase plays a role in protein targeting (17Forgac M. FEBS Lett. 1998; 440: 258-263Crossref PubMed Scopus (118) Google Scholar). Inhibition of the V-ATPase with the drug bafilomycin A1, as well as V-ATPase null mutations, resulted in the accumulation and missorting of precursor forms of various vacuolar proteins (18Banta L.M. Robinson J.S. Klionsky D.J. Emr S.D. J. Cell Biol. 1988; 107: 1369-1383Crossref PubMed Scopus (299) Google Scholar, 19Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (145) Google Scholar, 20Umemoto N. Yoshihisa T. Hirata R. Anraku Y. J. Biol. Chem. 1990; 265: 18447-18453Abstract Full Text PDF PubMed Google Scholar, 21Klionsky D.J. Nelson H. Nelson N. J. Biol. Chem. 1992; 267: 3416-3422Abstract Full Text PDF PubMed Google Scholar, 22Yaver D.S. Nelson H. Nelson N. Klionsky D.J. J. Biol. Chem. 1993; 268: 10564-10572Abstract Full Text PDF PubMed Google Scholar). In addition, it was shown that, under those conditions, precursor forms of vacuolar hydrolases were accumulated within the Golgi complex and/or post-Golgi compartments (22Yaver D.S. Nelson H. Nelson N. Klionsky D.J. J. Biol. Chem. 1993; 268: 10564-10572Abstract Full Text PDF PubMed Google Scholar) and that proteins having luminally oriented targeting signals were directly dependent on compartment acidification for efficient vacuolar delivery (23Morano K.A Klionsky D.J. J. Cell Sci. 1994; 107: 2813-2824Crossref PubMed Google Scholar). Studies with different vacuolar proteins revealed involvement of at least two distinct targeting pathways to the vacuole (24Wendland B. Emr S.D. Riezman H. Curr. Opin. Cell Biol. 1998; 10: 513-522Crossref PubMed Scopus (150) Google Scholar). One goes through the endosomes, and the other delivers the proteins directly to the vacuole from the post-Golgi vesicles. Similarly, multiple delivery pathways of plasma membrane proteins may exist (25de Kerchove d'Exaerde A. Supply P. Goffeau A. Yeast. 1996; 12: 907-916Crossref PubMed Scopus (30) Google Scholar). Studies of Pma1p sorting revealed involvement of several proteins needed specifically for its transport to the plasma membrane (9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 26Luo W.J. Chang A. J. Cell Biol. 1997; 138: 731-746Crossref PubMed Scopus (91) Google Scholar, 27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). In this work, we report on such a differential effect of V-ATPase null mutations on the sorting of Pma1p to the plasma membrane. We show that while the targeting of Pma1p is highly affected by the V-ATPase null mutations, these mutations have very little effect on the targeting of Gas1p, Sec61p, or Sed5p into their respective compartments. Until now, every V-ATPase mutant studied that abolished the activity of the enzyme shared more then one feature with the other V-ATPase mutants' phenotypes. Since medium pH growth requirements are common to a variety of different mutations, in order to characterize a new V-ATPase-defective mutant, we have to demonstrate several features that coexist with and are similar to a known subunit of the V-ATPase-deleted mutant. The sorting profile of Pma1p in the V-ATPase mutants can be added to this assortment of features. Kex2p and Gef1p, two membrane proteins residing in the post-Golgi vesicles, were indicated as influencing V-ATPase activity. Their null mutants were created in two different yeast strains, and both had intact V-ATPase activity and regular quinacrine accumulation in the vacuole as well as wild-type profile of Pma1p distribution. The conclusions from these observations are that neither Kex2p nor Gef1p has a general effect upon the V-ATPase activity; nor do they affect the sorting path of Pma1p in the secretory pathway in the way that the V-ATPase null mutant does. DISCUSSIONPma1p is an essential enzyme that provides the protonmotive force for the yeast plasma membrane and plays a major role in the pH homeostasis of the cell. The amount of Pma1p in the plasma membrane is also under strict control, and the enzyme is present usually in a relatively constant amount (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). The V-ATPase complements the function of Pma1p in organelles and membranes of the vacuolar system (52Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar). In addition to its function as the main energizing enzyme in the vacuolar system, the V-ATPase was found to be involved in the sorting processes of proteins to the vacuolar compartment in yeast (53Nelson N. Klionsky D.J. Experientia. 1996; 52: 1101-1110Crossref PubMed Scopus (33) Google Scholar). It has long been known that acidification of a lysosomal compartment is required for proper sorting of soluble lysosomal proteases in mammalian cells, utilizing the mannose 6-phosphate receptor pathway (54Mellman I. J. Exp. Biol. 1992; 172: 39-45Crossref PubMed Google Scholar, 55Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1331) Google Scholar). The pre-Golgi and cis-Golgi membranes were also shown to contain a V-ATPase activity. There it regulates retrograde membrane traffic at the ER-Golgi pathway (56Palokangas H. Ying M. Vaananen K. Saraste J. Mol. Biol. Cell. 1998; 9: 3561-3578Crossref PubMed Scopus (86) Google Scholar). A compartment acidification requirement was demonstrated in the targeting of GPP130 (Golgiphosphoprotein of 130 kDa) to the early Golgi (57Linstedt A.D. Mehta A. Suhan J. Reggio H. Hauri H.P. Mol. Biol. Cell. 1997; 8: 1073-1087Crossref PubMed Scopus (99) Google Scholar). It would be expected that the other secretory sites would also be sensitive to the pH in their respective compartments.In this study, we examined the influence of V-ATPase null mutations in yeast on the processes of targeting to the plasma membrane by following their effect on the Pma1p distribution. Numerous studies investigated the influence of deletion or overexpression of proteins, known to reside in the secretory pathway, on Pma1p sorting and targeting to the plasma membrane in yeast. Among them, Ast1p, Ast2p, Vps8p, and Vps36p were investigated and found to influence the Pma1p distribution (9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 58Luo W.J. Chang A. Mol. Biol. Cell. 2000; 11: 579-592Crossref PubMed Scopus (63) Google Scholar). By comparison with the Gas1p, another plasma membrane resident, several proteins with a differential effect on Pma1p were discovered. Among them is Lst1p, which was suggested to take part in the transport of Pma1p from ER to the Golgi compartment (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). Another was identified as Eps1p (ER-retained PMA1suppressor), which functions in ER quality control (45Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). The mutant of Eps1p suppresses the D378N phenotype by allowing both the mutant and wild-type Pma1p molecules to travel to the plasma membrane. Its null mutant does not affect the sorting of native Pma1p at all, whereas it causes a marked accumulation of the 105-kDa ER form of Gas1p in the ER.We found that V-ATPase null mutants have a specific effect on Pma1p as well. To determine its distribution on the sucrose gradient, we used Sec61p as a marker of the ER, Sed5p as a marker for the Golgi compartment, and Gas1p as the other plasma membrane marker. There was no change in the distribution of Sec61p and Sed5p in the mutant in comparison with the wild-type strain; nor was the amount of Gas1p in the plasma membrane reduced, although its 105-kDa intermediate was elevated in the ER in comparison with the wild-type strain. However, Pma1p amounts in the plasma membrane in the ATPase-depleted mutants were markedly reduced, and a large amount of the protein was accumulated in the ER in a nonactive form.The fact that, in V-ATPase null mutants, the distribution of Gas1p differs from Pma1p suggests that lack of acidification of the various compartments differentially changes the protein composition of their membranes but not the characteristics of the membranes in general. The decline in Pma1p in plasma membrane may be a unique feature of this protein, whereas the slowdown of movement from ER to Golgi, as demonstrated by Gas1p in the mutant yeast, might be a general feature of the mutant.The question remains as to the site of the crucial secretory pathway compartment, whose acidification by V-ATPase is vital. The fact that the site of Pma1p accumulation in the mutant is in the ER suggests that the passage from ER to Golgi in the V-ATPase mutants is impaired. This hypothesis is supported by several previous results. It was shown in mammalian cells that retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the V-ATPase-specific inhibitor Bafilomycin A1 (56Palokangas H. Ying M. Vaananen K. Saraste J. Mol. Biol. Cell. 1998; 9: 3561-3578Crossref PubMed Scopus (86) Google Scholar). In yeast, the deletion of the LST1 gene that encodes for a peripheral ER membrane protein results in a profile of Pma1p distribution on sucrose gradient similar to the one of V-ATPase null mutants (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). It was suggested that Lst1p takes part in efficient packaging of Pma1p into vesicles derived from the ER. Moreover, it was shown that the plasma membrane H+-ATPase undergoes pH-dependent conformational changes (59Blanpain J.P. Ronjat M. Supply P. Dufour J.P. Goffeau A. Dupont Y. J. Biol. Chem. 1992; 267: 3735-3740Abstract Full Text PDF PubMed Google Scholar). If we assume that the V-ATPase functions incis-Golgi, then the impaired sorting of Pma1p in the V-ATPase null mutant could be explained by damage to the retrograde transport of factors such as Lst1p or conformational changes in Pma1p, imposed by the improper pH conditions.The defect that V-ATPase null mutants are mostly checked for is their inability to grow on a buffered pH 7.5 medium. However, these mutants are also defective in growth on a low pH medium (13Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar). The inability to grow on very acidic medium was demonstrated for a wide range of Pma1p mutants as well and was explained by a low activity of the mutant, stemming from either the mutation or competition of the mutant on the targeting to the membrane with the intact protein (12McCusker J.H. Perlin D.S. Haber J.E. Mol. Cell. Biol. 1987; 7: 4082-4088Crossref PubMed Scopus (138) Google Scholar, 60Wang G. Tamas M.J. Hall M.J. Pascual-Ahuir A. Perlin D.S. J. Biol. Chem. 1996; 271: 25438-25445Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Since we find that in V-ATPase null mutants the amount of Pma1p in the plasma membrane is markedly reduced, it is tempting to suggest that the reduced growth rate at low pH is caused by reduced levels of Pma1p in the plasma membrane of those mutants.In this study, we show that the altered distribution of Pma1p in V-ATPase null mutant can serve as another characteristic feature of it. Kex2p and Gef1p were both suggested to be involved in the proper function of the acidification by the V-ATPase (48Oluwatosin Y.E. Kane P.M. Mol. Cell. Biol. 1998; 18: 1534-1543Crossref PubMed Scopus (30) Google Scholar, 49Gaxiola R.A. Yuan D.S. Klausner R.D. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4046-4050Crossref PubMed Scopus (148) Google Scholar). In the case of the ΔKEX2 null mutant, there has been a discrepancy in the original paper, where the ATPase and proton pumping activities in isolated vacuoles of this mutant were undamaged, whereas the vacuoles failed to accumulate quinacrine. To exclude the possibility that in our strain (W303), uniquely, the deletion of Kex2p did not affect the quinacrine accumulation in the vacuoles, we disrupted this gene in two additional strains and in them observed as well that the ΔKEX2 mutant was fully active in quinacrine accumulation into their vacuoles. In addition, when we checked the distribution of the Pma1p in theΔKEX2 and ΔGEF1mutants, it was similar to the wild type and not to the V-ATPase mutant. By metal supplementation in the growth medium, we showed that the addition of 5 μm Cu2+ rescued theΔKEX2 and ΔGEF1 mutants and enabled their growth on pH 7.5 medium, whereas it did not have any effect upon the V-ATPase mutant. Therefore, a direct interaction between Kex2p or Gef1p and V-ATPase can be excluded. However, their activity may be involved in some cellular functions, such as anion transport or metal ion homeostasis, in specific organelles of the secretory pathway that also affect the growth at high pH levels in the medium. Pma1p is an essential enzyme that provides the protonmotive force for the yeast plasma membrane and plays a major role in the pH homeostasis of the cell. The amount of Pma1p in the plasma membrane is also under strict control, and the enzyme is present usually in a relatively constant amount (4Serrano R. FEBS Lett. 1993; 325: 108-111Crossref PubMed Scopus (43) Google Scholar). The V-ATPase complements the function of Pma1p in organelles and membranes of the vacuolar system (52Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar). In addition to its function as the main energizing enzyme in the vacuolar system, the V-ATPase was found to be involved in the sorting processes of proteins to the vacuolar compartment in yeast (53Nelson N. Klionsky D.J. Experientia. 1996; 52: 1101-1110Crossref PubMed Scopus (33) Google Scholar). It has long been known that acidification of a lysosomal compartment is required for proper sorting of soluble lysosomal proteases in mammalian cells, utilizing the mannose 6-phosphate receptor pathway (54Mellman I. J. Exp. Biol. 1992; 172: 39-45Crossref PubMed Google Scholar, 55Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1331) Google Scholar). The pre-Golgi and cis-Golgi membranes were also shown to contain a V-ATPase activity. There it regulates retrograde membrane traffic at the ER-Golgi pathway (56Palokangas H. Ying M. Vaananen K. Saraste J. Mol. Biol. Cell. 1998; 9: 3561-3578Crossref PubMed Scopus (86) Google Scholar). A compartment acidification requirement was demonstrated in the targeting of GPP130 (Golgiphosphoprotein of 130 kDa) to the early Golgi (57Linstedt A.D. Mehta A. Suhan J. Reggio H. Hauri H.P. Mol. Biol. Cell. 1997; 8: 1073-1087Crossref PubMed Scopus (99) Google Scholar). It would be expected that the other secretory sites would also be sensitive to the pH in their respective compartments. In this study, we examined the influence of V-ATPase null mutations in yeast on the processes of targeting to the plasma membrane by following their effect on the Pma1p distribution. Numerous studies investigated the influence of deletion or overexpression of proteins, known to reside in the secretory pathway, on Pma1p sorting and targeting to the plasma membrane in yeast. Among them, Ast1p, Ast2p, Vps8p, and Vps36p were investigated and found to influence the Pma1p distribution (9Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar, 58Luo W.J. Chang A. Mol. Biol. Cell. 2000; 11: 579-592Crossref PubMed Scopus (63) Google Scholar). By comparison with the Gas1p, another plasma membrane resident, several proteins with a differential effect on Pma1p were discovered. Among them is Lst1p, which was suggested to take part in the transport of Pma1p from ER to the Golgi compartment (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). Another was identified as Eps1p (ER-retained PMA1suppressor), which functions in ER quality control (45Wang Q. Chang A. EMBO J. 1999; 18: 5972-5982Crossref PubMed Scopus (88) Google Scholar). The mutant of Eps1p suppresses the D378N phenotype by allowing both the mutant and wild-type Pma1p molecules to travel to the plasma membrane. Its null mutant does not affect the sorting of native Pma1p at all, whereas it causes a marked accumulation of the 105-kDa ER form of Gas1p in the ER. We found that V-ATPase null mutants have a specific effect on Pma1p as well. To determine its distribution on the sucrose gradient, we used Sec61p as a marker of the ER, Sed5p as a marker for the Golgi compartment, and Gas1p as the other plasma membrane marker. There was no change in the distribution of Sec61p and Sed5p in the mutant in comparison with the wild-type strain; nor was the amount of Gas1p in the plasma membrane reduced, although its 105-kDa intermediate was elevated in the ER in comparison with the wild-type strain. However, Pma1p amounts in the plasma membrane in the ATPase-depleted mutants were markedly reduced, and a large amount of the protein was accumulated in the ER in a nonactive form. The fact that, in V-ATPase null mutants, the distribution of Gas1p differs from Pma1p suggests that lack of acidification of the various compartments differentially changes the protein composition of their membranes but not the characteristics of the membranes in general. The decline in Pma1p in plasma membrane may be a unique feature of this protein, whereas the slowdown of movement from ER to Golgi, as demonstrated by Gas1p in the mutant yeast, might be a general feature of the mutant. The question remains as to the site of the crucial secretory pathway compartment, whose acidification by V-ATPase is vital. The fact that the site of Pma1p accumulation in the mutant is in the ER suggests that the passage from ER to Golgi in the V-ATPase mutants is impaired. This hypothesis is supported by several previous results. It was shown in mammalian cells that retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the V-ATPase-specific inhibitor Bafilomycin A1 (56Palokangas H. Ying M. Vaananen K. Saraste J. Mol. Biol. Cell. 1998; 9: 3561-3578Crossref PubMed Scopus (86) Google Scholar). In yeast, the deletion of the LST1 gene that encodes for a peripheral ER membrane protein results in a profile of Pma1p distribution on sucrose gradient similar to the one of V-ATPase null mutants (27Roberg K.J. Crotwell M. Espenshade P. Gimeno R. Kaiser C.A. J. Cell Biol. 1999; 145: 659-672Crossref PubMed Scopus (128) Google Scholar). It was suggested that Lst1p takes part in efficient packaging of Pma1p into vesicles derived from the ER. Moreover, it was shown that the plasma membrane H+-ATPase undergoes pH-dependent conformational changes (59Blanpain J.P. Ronjat M. Supply P. Dufour J.P. Goffeau A. Dupont Y. J. Biol. Chem. 1992; 267: 3735-3740Abstract Full Text PDF PubMed Google Scholar). If we assume that the V-ATPase functions incis-Golgi, then the impaired sorting of Pma1p in the V-ATPase null mutant could be explained by damage to the retrograde transport of factors such as Lst1p or conformational changes in Pma1p, imposed by the improper pH conditions. The defect that V-ATPase null mutants are mostly checked for is their inability to grow on a buffered pH 7.5 medium. However, these mutants are also defective in growth on a low pH medium (13Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar). The inability to grow on very acidic medium was demonstrated for a wide range of Pma1p mutants as well and was explained by a low activity of the mutant, stemming from either the mutation or competition of the mutant on the targeting to the membrane with the intact protein (12McCusker J.H. Perlin D.S. Haber J.E. Mol. Cell. Biol. 1987; 7: 4082-4088Crossref PubMed Scopus (138) Google Scholar, 60Wang G. Tamas M.J. Hall M.J. Pascual-Ahuir A. Perlin D.S. J. Biol. Chem. 1996; 271: 25438-25445Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Since we find that in V-ATPase null mutants the amount of Pma1p in the plasma membrane is markedly reduced, it is tempting to suggest that the reduced growth rate at low pH is caused by reduced levels of Pma1p in the plasma membrane of those mutants. In this study, we show that the altered distribution of Pma1p in V-ATPase null mutant can serve as another characteristic feature of it. Kex2p and Gef1p were both suggested to be involved in the proper function of the acidification by the V-ATPase (48Oluwatosin Y.E. Kane P.M. Mol. Cell. Biol. 1998; 18: 1534-1543Crossref PubMed Scopus (30) Google Scholar, 49Gaxiola R.A. Yuan D.S. Klausner R.D. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4046-4050Crossref PubMed Scopus (148) Google Scholar). In the case of the ΔKEX2 null mutant, there has been a discrepancy in the original paper, where the ATPase and proton pumping activities in isolated vacuoles of this mutant were undamaged, whereas the vacuoles failed to accumulate quinacrine. To exclude the possibility that in our strain (W303), uniquely, the deletion of Kex2p did not affect the quinacrine accumulation in the vacuoles, we disrupted this gene in two additional strains and in them observed as well that the ΔKEX2 mutant was fully active in quinacrine accumulation into their vacuoles. In addition, when we checked the distribution of the Pma1p in theΔKEX2 and ΔGEF1mutants, it was similar to the wild type and not to the V-ATPase mutant. By metal supplementation in the growth medium, we showed that the addition of 5 μm Cu2+ rescued theΔKEX2 and ΔGEF1 mutants and enabled their growth on pH 7.5 medium, whereas it did not have any effect upon the V-ATPase mutant. Therefore, a direct interaction between Kex2p or Gef1p and V-ATPase can be excluded. However, their activity may be involved in some cellular functions, such as anion transport or metal ion homeostasis, in specific organelles of the secretory pathway that also affect the growth at high pH levels in the medium. We thank Drs. Randy Schekman and Howard Riezman for providing yeast strains and antibodies." @default.
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W1999036031 title "Altered Distribution of the Yeast Plasma Membrane H+-ATPase as a Feature of Vacuolar H+-ATPase Null Mutants" @default.
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