A bovine cDNA and a yeast gene (VMA8) encoding the subunit D of the vacuolar H(+)-ATPase.
Subunit D of vacuolar H(+)-ATPase (V-ATPase) from bovine chromaffin granules was subjected to partial proteolysis and amino acid sequencing. A cDNA encoding this subunit was isolated and sequenced. The predicted open reading frame encodes a protein of 247 amino acids with a calculated molecular weight of 28,336. Northern blot analysis revealed an mRNA distribution with higher transcript amounts in tissues that are active in secretion. A homologous gene was identified as open reading frame 11 in chromosome V of Saccharomyces cerevisiae. The two proteins exhibit 55% identity with several conservative replacements. Interruption of the yeast gene, denoted as VMA8, resulted in the null mutant delta vma8::URA3 that, like all the other V-ATPase null mutants, did not grow on medium buffered at pH 7.5 and showed no accumulation of quinacrine into their vacuoles. Transformation of the null mutant with a plasmid containing the VMA8 gene restored the wild-type phenotype. This supports the conclusion that subunit D is an integral subunit of the catalytic sector of V-ATPase and its structural analysis suggests analogy to the gamma subunit of F-ATPases.
Nelson
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Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Mandiyan
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Nelson
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D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000737:chemistry; D010641:Phenotype; D011506:Proteins / Q000235:genetics*; D006180:Proton-Translocating ATPases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*
D018076:DNA, Complementary; D010446:Peptide Fragments; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C091579:VMA8 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases