Novel vacuolar H+-ATPase complexes resulting from overproduction of Vma5p and Vma13p.
The vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex composed of two sectors: V(1), a peripheral membrane sector responsible for ATP hydrolysis, and V(0), an integral membrane sector that forms a proton pore. Vma5p and Vma13p are V(1) sector subunits that have been implicated in the structural and functional coupling of the V-ATPase. Cells overexpressing Vma5p and Vma13p demonstrate a classic Vma(-) growth phenotype. Closer biochemical examination of Vma13p-overproducing strains revealed a functionally uncoupled V-ATPase in vacuolar vesicles. The ATP hydrolysis rate was 72% of the wild-type rate; but there was no proton translocation, and two V(1) subunits (Vma4p and Vma8p) were present at lower levels. Vma5p overproduction moderately affected both V-ATPase activity and proton translocation without affecting enzyme assembly. High level overexpression of Vma5p and Vma13p was lethal even in wild-type cells. In the absence of an intact V(0) sector, overproduction of Vma5p and Vma13p had a more detrimental effect on growth than their deletion. Overproduced Vma5p associated with cytosolic V(1) complexes; this association may cause the lethality.
Keenan Curtis
Kelly
K
Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA.
Kane
Patricia M
PM
D000255:Adenosine Triphosphate / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000201:enzymology; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014170:Transformation, Genetic; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C447617:VMA13 protein, S cerevisiae; C447616:VMA5 protein, S cerevisiae; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases