A novel mechanism for regulation of vacuolar acidification.
We have recently demonstrated that Cys-254 of the 73-kDa A subunit of the clathrin-coated vesicle (H+)-ATPase is responsible for sensitivity of the enzyme to sulfhydryl reagents (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). In the present study we observe that for the purified enzyme, disulfide bond formation causes inactivation of proton transport which is reversed by dithiothreitol (DTT). DTT also restores activity of the oxidized enzyme following treatment with N-ethylmaleimide (NEM). These results indicate that disulfide bond formation between the NEM-reactive cysteine (Cys-254) and a closely proximal cysteine residue leads to inactivation of the (H+)-ATPase. To test whether sulfhydryl-disulfide bond interchange may play a role in regulating vacuolar acidification in vivo, we have determined what fraction of the (H+)-ATPase is disulfide-bonded in native clathrin-coated vesicles. Vesicles were isolated under conditions that prevent any change in the oxidation state of the sulfhydryl groups. NEM treatment of vesicles causes nearly complete loss of activity while subsequent treatment with DTT restores 50% of the activity of the fully reduced vesicles. By contrast, treatment of fully reduced vesicles with NEM leads to inactivation which is not reversed by DTT. These results indicate that a significant fraction of the clathrin-coated vesicle (H+)-ATPase exists in an inactive, disulfide-bonded state and suggest that sulfhydryl-disulfide bond interconversion may play a role in controlling vacuolar (H+)-ATPase (V-ATPase) activity in vivo.
Feng
Y
Y
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Forgac
M
M
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000502:physiology*
D002966:Clathrin; D004220:Disulfides; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol