A genomic screen for yeast vacuolar membrane ATPase mutants.
V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.
Sambade
Maria
M
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
Alba
Mercedes
M
Smardon
Anne M
AM
West
Robert W
RW
Kane
Patricia M
PM
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000032:analysis; D019703:Calcineurin / Q000378:metabolism; D017353:Gene Deletion; D016681:Genome, Fungal; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D009154:Mutation; D011796:Quinacrine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D000469:Alkaline Phosphatase; D019703:Calcineurin; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D011796:Quinacrine