A Novel Family of Yeast Chaperons Involved in the Distribution of V-ATPase and Other Membrane Proteins

Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar TransporterChaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (ΔVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H+-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of ΔVTC1 and ΔVMA subunits. Null mutations in genes encoding V-ATPase subunits in Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethylmethanesulfonate causes mutations that suppress the V-ATPase null phenotype, and the mutant cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (suppressor of V-ATPase function). The frequency of svf is relatively high, suggesting a large target containing several genes for the ethylmethanesulfonate mutagenesis. The suppressors' frequency is dependent on the individual genes that were inactivated to manifest the V-ATPase null mutation. The svf mutations are recessive, because crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that are unable to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or sensitivity and distribution of membrane proteins in different targets was discovered. The family was defined as VTC (Vacuolar TransporterChaperon) and it contains four genes in the S. cerevisiae genome. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in svf phenotype manifested by growth at pH 7.5. Deletion of the VTC1 gene (ΔVTC1) results in a reduced amount of V-ATPase in the vacuolar membrane. These mutant cells fail to accumulate quinacrine into their vacuoles, but they are able to grow at pH 7.5. The VTC1 null mutant also results in a reduced amount of the plasma membrane H+-ATPase (Pma1p) in membrane preparations and possibly mis-targeting. This observation may provide an explanation for the svf phenotype in the double disruptant mutants of ΔVTC1 and ΔVMA subunits. vacuolar H+-ATPase endoplasmic reticulum 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid polymerase chain reaction ethylmethanesulfonate Null mutations in genes encoding vacuolar H+-ATPase (V-ATPase)1 subunits are likely to be lethal for most eukaryotic cells, because energization of the vacuolar system by this enzyme drives vital secondary transport processes across membranes of vacuolar-derived organelles (1Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar, 2Nelson N. Klionsky D.J. Experientia. 1996; 52: 1101-1110Crossref PubMed Scopus (34) Google Scholar). Disruption of genes encoding V-ATPase subunits in Neurospora and Drosophila melanogaster caused lethality (3Bowman E.J. O'Neill F.J. Bowman B.J. J. Biol. 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Biol. 1997; PubMed Scopus Google that of to were and the was for cells were in of to at The cells on for and of the cells by and in containing and The was at and on for The cells were by and in of The cells were with the and in of the of the was on the with of low that is at and with a of quinacrine into the was by with at and a of a growth of V-ATPase null mutants on at pH N. Nelson H. Nelson N. Scholar). were able to grow the the V-ATPase null mutants are by the of most of the of their it is that the the V-ATPase and of the enzyme N. Nelson H. Nelson N. Scholar). with quinacrine by these mutants and into that their V-ATPase is not N. Nelson H. Nelson N. Scholar). The mutants were denoted as svf mutants (suppressor of V-ATPase function). The large of that at pH a with subunits as the target for mutagenesis. that svf are A mutant that can grow at pH was with the null mutant that not grow at pH 7.5. strains of V-ATPase null mutants that the and svf were not able to grow on at pH 7.5. is but not that the results in the of a that is of a large The of was by crossing the of svf mutants of as as of diploid strains of at pH and of diploid strains of at pH 7.5. This indicates that several are involved in the of svf suggesting the of several gene in the V-ATPase null mutants to high the mutation by the inactivated a which resulted in growth at pH the V-ATPase was The of one copy of the gene in diploid cells the and resulted in growth on a medium at pH 7.5. V-ATPase is of several the frequency of svf in V-ATPase null mutants in which different genes encoding subunits of the enzyme were in of V-ATPase null mutants in which genes encoding subunits and of the were to higher of svf of V-ATPase null mutants in membrane subunits resulted in frequency of This also a that was to be for the of the membrane T.H. Forgac M. Annu. Rev. Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar, T.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, H. S. Nelson N. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). is that null mutants in and which are of the membrane frequency of svf and the for which not the of the of the subunits T.H. Biol. 1994; PubMed Scopus Google Scholar, R. Graham A. T.H. Anraku Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google Scholar). was to that the double mutant ΔVTC1 at pH 7.5. this a diploid containing one of and one of ΔVTC1 was This diploid a that the of the four strains resulted from of one The and genes were by and as of the and to and the other to and was for but that were from this at pH but to accumulate quinacrine into their was for VTC1 but and cells to grow at pH and to accumulate to a double disruptant mutant in genes and The cells that were from this double mutant were able to grow at pH and as to accumulate quinacrine into their a phenotype in all the The that of VTC1 in which genes encoding V-ATPase were able to grow at pH that VTC1 is one for svf mutants a diploid containing of and one of ΔVTC1 to grow at pH that the of VTC1 resulted in a svf strains of which were for the of were with the VTC1 The were for growth at pH 7.5. svf to grow on at pH suggesting that this svf resulted from of VTC1 VTC1 encode a of with A in the yeast genome with the homologous genes that were and These genes encode proteins of and and a large in their and a with to in their A with or an homologous gene of which is the and to the of and the of the acid of the four of the VTC gene the it was to it is present in the as a with the yeast membranes and is not present in the as a that not be in the ΔVTC1 or in the double disruptant mutant disruption of each of the genes on the of These results that and as a which the of and evidence that for cellular by the of of by in different VTC disruptant The was in by a containing with a relatively acid for and The yeast strains were in medium at pH to an of The cells were by by and were as and The cells were by with and the membranes were by for and for of were in each and the and was as and A in the a large family of proteins with but at the to the S. the the proteins and and the and and are involved in is to the low at the by into the to the N. 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Rev. 1997; PubMed Google Scholar). for their transport A in homologous proteins in D. and a in and The gene from C. was denoted as an in The gene is and as to the yeast the contains a and with the with the The Drosophila gene is present on with The high to the C. in and acid the of these proteins is not in to their they are likely to a similar of with this a acid of was in all of a of of the proteins at the of the The disruption of VTC genes resulted in different V-ATPase as by quinacrine and into the of V-ATPase subunits were by that the disruption of VTC1 resulted in of the of subunits and in the This is in with the reduced of the The disruption of other VTC genes not the of these subunits. reduced of from The of and was at at the ΔVTC1 the V-ATPase null phenotype, it is that the may result from in the distribution of different in the yeast in the amount of was also reduced in membrane of ΔVTC1 in to and other were to in distribution of in membranes of the in the distribution of in the of the membrane was not in ΔVTC1 in with the of the that the of in ΔVTC1 are than in membrane preparations of the of ΔVTC1 with a VTC1 gene the of Null mutations in V-ATPase subunits result in of vacuolar proteins N. 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