The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H(+)-ATPase.
Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1.2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25,286 Daltons, has two distinct membrane-spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H(+)-ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM-CaCl2 or 4 mM-ZnCl2 or buffered to pH 7.5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H(+)-ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).
Bachhawat
A K
AK
Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA 15213.
Manolson
M F
MF
Murdock
D G
DG
Garman
J D
JD
Jones
E W
EW
D000469:Alkaline Phosphatase / Q000032:analysis; D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics*; D005816:Genetic Complementation Test; D006867:Hydrolases / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010641:Phenotype; D017433:Protein Structure, Secondary; D017494:Proton Pumps; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000201:enzymology; D014617:Vacuoles / Q000201:enzymology*
D005656:Fungal Proteins; D017494:Proton Pumps; D006867:Hydrolases; D000469:Alkaline Phosphatase