Isolation of vacuolar membrane H(+)-ATPase-deficient yeast mutants; the VMA5 and VMA4 genes are essential for assembly and activity of the vacuolar H(+)-ATPase.
The vacuolar membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae is a multisubunit enzyme complex composed of an integral membrane V0 sector, and a peripherally associated V1 sector. Deletion of one of several structural genes for vacuolar H(+)-ATPase subunits was previously demonstrated to prevent proper assembly of the remaining V1 subunits onto the vacuolar membrane (Kane, P.M., Kuehn, M.C., Howald-Stevenson, I., and Stevens, T.H. (1992) J. Biol. Chem. 267, 447-454). A genetic screen was designed to identify new genes whose products were essential for the synthesis, assembly, and/or function of the yeast vacuolar H(+)-ATPase. Mutants were identified based on phenotypes associated with vacuolar membrane H(+)-ATPase loss of function (vma), including an inability to grow on media buffered at neutral pH. Representatives in five complementation groups were identified, including four novel mutant vma5, vma21, vma22, and vma23, all of which were defective in vacuolar ATPase enzyme activity. We report here the characterization of two genes, VMA4 and VMA5, that encode peripheral subunits of the vacuolar H(+)-ATPase. We determined that VMA5 encodes the 42-kDa subunit of the vacuolar H(+)-ATPase. The VMA4 gene, originally described by Foury (Foury, F. (1990) J. Biol. Chem. 265, 18554-18560), was determined to encode the 27-kDa subunit of the purified yeast vacuolar H(+)-ATPase. Characterization of the vma5 and vma4 mutants revealed that the 42- and 27-kDa subunits are essential for the assembly of the peripheral membrane portion of the H(+)-ATPase onto the vacuolar membrane.
Ho
M N
MN
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Hill
K J
KJ
Lindorfer
M A
MA
Stevens
T H
TH
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D007425:Intracellular Membranes / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D014617:Vacuoles / Q000201:enzymology*
D004271:DNA, Fungal; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases