Evidence for a conserved 95–120 KDA subunit associated with and essential for activity of V-ATPases

Abstract

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.

Self-Assembled Nanonematicide Induces Adverse Effects on Oxidative Stress, Succinate Dehydrogenase Activity, and ATP Generation

“…The upregulation of these genes revealed the enhanced uptake of flu nanoagents by nematodes via the endocytic pathway. This result is in line with our previous discoveries, indicating that SPc can trigger clathrin-mediated endocytosis to facilitate the uptake of diverse exogenous materials. , Among the encoded proteins of energy-related genes, vATPA (V-type proton ATPase subunit A) is a multisubunit enzyme that hydrolyzes ATP and a membrane integral complex . The downregulation of the vATPA gene suggested that the flu nanoagent might adversely influence ATP generation compared to flu alone, which further disturbed the expression of other energy-related genes.…”

“…23,24 Among the encoded proteins of energy-related genes, vATPA (V-type proton ATPase subunit A) is a multisubunit enzyme that hydrolyzes ATP and a membrane integral complex. 71 The downregulation of the vATPA gene suggested that the flu nanoagent might adversely influence ATP generation compared to flu alone, which further disturbed the expression of other energy-related genes. For instance, the PIF1 (ATP-dependent DNA helicase PIF1) gene, which maintains both mitochondrial and nuclear genome stability, and the Ath (ATP-dependent RNA helicase DHX33) gene, which coordinates germline stem cell differentiation, were upregulated.…”