Inhibition of vacuolar H(+)-ATPase by disulfide bond formation between cysteine 254 and cysteine 532 in subunit A.
We have previously demonstrated that the coated vesicle vacuolar H(+)-ATPase (V-ATPase) can be inactivated by formation of intramolecular disulfide bonds (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 19769-19772). The disulfide bond responsible for inactivation can be distinguished from other disulfide bonds that form by the fact that formation of the inactivating disulfide bond is blocked by ATP or high ionic strength. By taking advantage of these properties, we selectively labeled the ATPase at the relevant cysteine residues with fluorescein maleimide. After analyzing the proteolytic fragments that contain the labeled cysteine residues, we found that cysteine 254 and cysteine 532 in subunit A of the bovine V-ATPase are the residues that form the disulfide bond resulting in inactivation of the enzyme. Cysteine 254 and cysteine 532 correspond to 2 of the 3 cysteine residues that are conserved in all available V-ATPase A subunit sequences. Cysteine 254 is located in the consensus motif, G(X)4GKT, corresponding to residues 250-257, which is conserved in many nucleotide binding proteins. Cysteine 532 is located in a region not previously shown to be in proximity to the nucleotide binding site. Modification of cysteine 254 by disulfide bond formation with cysteine 532 or thio-disulfide exchange with cystine does not impair binding of 2-azido-[32P]ATP to the A subunit. The inhibition is therefore likely caused by disruption of the catalytic function of the ATPase on formation of the disulfide bond. A possible role in regulating intracellular acidification by reversible sulfhydryl oxidation and reduction is discussed.
Feng
Y
Y
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Forgac
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005033:Ethylmaleimide; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
D004220:Disulfides; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide