Evidence for a conserved 95-120 kDa subunit associated with and essential for activity of V-ATPases.
Vacuoles purified from Saccharomyces cerevisiae bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. Furthermore, the vacuolar H(+)-ATPase (V-ATPase) nucleotide binding subunits were no longer associated with vacuolar membranes yet were present at wild-type levels in yeast whole-cell extracts. The VPH1 gene was cloned by screening a lambda gt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vph1-1 mutation. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is required for vacuolar H(+)-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases. VPH1 encodes a predicted polypeptide of 840 amino acid residues (95.6 kDa) with putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with V-ATPase activity. Vph1p has 42% identity to the 116 kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, 42% identity to the TJ6 mouse immune suppressor factor, 42% identity to the Caenorhabditis elegans proton pump homologue and 54% identity to the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene.
Manolson
M F
MF
Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA 15213.
Proteau
D
D
Jones
E W
EW
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D029742:Caenorhabditis elegans Proteins; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology
D029742:Caenorhabditis elegans Proteins; C506985:vha-11 protein, C elegans; C506986:vha-2 protein, C elegans; C506987:vha-3 protein, C elegans; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; C506981:unc-32 protein, C elegans