Novel Vacuolar H+-ATPase Complexes Resulting from Overproduction of Vma5p and Vma13p

Kelly Keenan Curtis, Patricia M. Kane
2001 Journal of Biological Chemistry  
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
more » ... ng 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. . 1 The abbreviations used are: V-ATPase, vacuolar H ϩ -ATPase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Mes, 4-morpholineethanesulfonic acid; SD-Ura, supplemented minimal medium containing 2% dextrose.
doi:10.1074/jbc.m107777200 pmid:11717306 fatcat:izqksen7a5hsnmwhj43nrzvjji