VMA12 is essential for assembly of the vacuolar H(+)-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae.
vma12 mutants of the yeast Saccharomyces cerevisiae, which were originally identified as calcium-sensitive (cls) mutants that were also respiratory deficient (Pet-), have a defect in vacuolar membrane H(+)-ATPase activity (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The VMA12 gene was cloned by complementation of the growth defects of vma12 mutants. The nucleotide sequence of the gene predicts a polypeptide of 215 amino acids (25.2 kDa) with two putative membrane-spanning domains. A null vma12 mutant, constructed by chromosomal deletion of the gene, is viable but has completely lost the vacuolar membrane H(+)-ATPase activity and exhibits the same growth defects as observed for the original vma12 mutants. Synthesis and targeting of the subunits of the H(+)-ATPase in the delta vma12 mutant cells were examined by Western blotting analyses of whole cell and vacuolar membrane protein extracts. None of the peripheral membrane subunits that we analyzed (the 69-, 60-, 42-, and 27-kDa subunits) was detected in the vacuolar membrane fractions, although the cellular levels of these polypeptides appeared to be normal. The 100- and 17-kDa integral membrane subunits of the enzyme were absent or present at a substantially reduced level in mutant vacuolar membrane fractions. Anti-Vma12p antibodies recognized a vacuolar protein with the expected molecular mass of 25 kDa. However, the Vma12 protein was not detected in the vacuolar membrane ATPase complex that had been solubilized with a zwitterionic detergent, ZW3-14, and purified by glycerol gradient centrifugation (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). These results indicate that the VMA12 gene product is not a component of the active vacuolar ATPase complex and instead suggest that this protein is required during the process of assembly and/or targeting of the enzyme complex to the vacuolar membrane.
Hirata
R
R
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Umemoto
N
N
Ho
M N
MN
Ohya
Y
Y
Stevens
T H
TH
Anraku
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000201:enzymology*
D004271:DNA, Fungal; D004274:DNA, Recombinant; D005656:Fungal Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D015345:Oligonucleotide Probes; D029701:Saccharomyces cerevisiae Proteins; C079033:VPH2 protein, S cerevisiae; D006180:Proton-Translocating ATPases