Clathrin-coated vesicles contain an ATP-dependent proton pump.

Publisher Website
Google Scholar
Abstract
Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+ and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a .DELTA.pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles. ATP-dependent [14C]methylamine uptake was abolished by the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and partially inhibited by the carboxyl reagent N,N''-dicyclohexylcarbodiimide but was unaffected by the Na+,K+-ATPase inhibitors strophanthidin (100 .mu.M) and vanadate (10 .mu.M) and the mitochondrial ATPase inhibitors oligomycin (10 .mu.g/ml) and aurovertin (1 .mu.g/ml). GTP, but not the nonhydrolyzable analog 5''-adenylyl imidodiphosphate, could support [14C]methylamine uptake. Dissipation of the membrane potential with K+ and valinomycin resulted in stimulation of [14C]methylamine uptake, whereas both FCCP and valinomycin stimulated the strophanthidin-resistant ATPase activity. These results are consistent with the existence of an electrogenic, ATP-dependent proton pump in clathrin-coated vesicles. This proton pump may play a role in the acidification events that are essential in receptor-mediated endocytosis.