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+-AT Pase (V-ATPase) can be inactivated by formation of intramolecular disu lfide bonds (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 19769 -19772). The disulfide bond responsible for inactivation can be distin guished from other disulfide bonds that form by the fact that formatio n of the inactivating disulfide bond is blocked by ATP or high ionic s trength. By taking advantage of these properties, we selectively label ed the ATPase at the relevant cysteine residues with fluorescein malei mide. After analyzing the proteolytic fragments that contain the label ed cysteine residues, we found that cysteine 254 and cysteine 532 in s ubunit A of the bovine V-ATPase are the residues that form the disulfi de bond resulting in inactivation of the enzyme. Cysteine 254 and cyst eine 532 correspond to 2 of the 3 cysteine residues that are conserved in all available V-ATPase A subunit sequences. Cysteine 254 is locate d in the consensus motif, G(X)(4)GKT, corresponding to residues 250-25 7, which is conserved in many nucleotide binding proteins. Cysteine 53 2 is located in a region not previously shown to be in proximity to th e nucleotide binding site. Modification of cysteine 254 by disulfide b ond formation with cysteine 532 or thio-disulfide exchange with cystin e does not impair binding of 2-azido[P-32]ATP to the A subunit. The in hibition is therefore likely caused by disruption of the catalytic fun ction of the ATPase on formation of the disulfide bond. A possible rol e in regulating intracellular acidification by reversible sulfhydryl o xidation and reduction is discussed.