Abstract
Ca(2+)-sensitive mutants of the yeast Saccharomyces cerevisiae showing a Pet- phenotype (cls7-cls11) have lesions in a system for maintaining intracellular Ca2+ homeostasis (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132, 979-988). Genetic and biochemical studies have demonstrated that these Pet- cls mutants are related to defects in vacuolar membrane H(+)-ATPase. CLS7 and CLS8 were found to be identical with the structural genes encoding subunit c (VMA3) and subunit a (VMA1), respectively, of the enzyme. In addition, these five mutants all had vma defects; no vacuolar membrane ATPase activity was detected in the cls cells, and the cls mutants showed a loss of ability to acidify the vacuole in vivo. Measurements of the cytosolic free Ca2+ concentration [( Ca2+]i) in individual cells showed that the average [Ca2+]i in wild-type cells was 150 +/- 80 nM, whereas that in five Pet- cls cells was 900 +/- 100 nM. These data are consistent with the observation that vacuolar membrane vesicles prepared from the Pet- cls cells have lost ATP-dependent Ca2+ uptake activities. The cls defects of vacuolar membrane H(+)-ATPase resulted in pleiotropic effects on several cellular activities, including Ca2+ homeostasis, glycerol metabolism, and phospholipid metabolism. The mutants showed an inositol-dependent phenotype, possibly due to alteration in regulation of phospholipid biosynthesis; the phosphatidylserine decarboxylase activities of the mutants were 15-50% of that of the wild-type cells and were not repressed by the addition of inositol. In contrast to the majority of previously isolated pet mutants (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225), the Pet- cls mutants showed no detectable mitochondrial defects. Taking all these findings into account, we suggest that at least six genes, VMA1 (CLS8, subunit a), VMA2 (subunit b), VMA3 (CLS7, subunit c), VMA11 (CLS9), VMA12 (CLS10), and VMA13 (CLS11), are required for expression of the vacuolar membrane H(+)-ATPase activity.
Highlights
These five mutants all had vma types, type IV mutants, which have normal calcium contents defects; no vacuolar membrane ATPase activity was but show increased initial ratesof Ca2+ uptake, have attracted detected in the cls cells, and thecls mutants showeda our attention because they all show a Pet- phenotype, and loss of ability to acidify thevacuole in vivo.Measure- this Pet- phenotypecosegregates with theCls- phenotype [1]
Wild-type cells was 150 2 80 nM, whereas that in five Sherman ( 2 ) first described pet mutants of S. cereuisiae, Pet- cls cells was 900 f 100 nM. These data are con- and in subsequent genetic studies, a number of pet mutants sistent with the observation that vacuolar membrane of 200 complementation groups have been obtained
These results clearly demonstrated that vma3 did not complement cls7 and una1 did not complement cls8; they suggest that CLS7 and CLSS are identical with VMA3 and VAMl, respectively. vma2 complemented any cls mutants (Fig. 3)
Summary
11 To whom correspondence should he addressed Dept.of Biology, thispaper.Miniprintis readwiththe aid of a standard. Cells were streaked on YPD, YPD 100 mM CaC12,and YP-glycerol plates and incubated for 2 days a t 30 "C. All cls and uma mutants grew on YPD, but not on YPD 100 mM CaC12 or YP-glycerol plates (see Fig. 2 and text). Cells died within 2 h (Fig. 1).The other Pet-cls mutants showed essentiallythesame growth properties(datanot shown). These resultssuggest that thecls defects havea lethal effect on the mutants in medium containing excess CaC12 and, in its absence, result in a lesion in metabolism of nonfermentable carbon sources (Table I).
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