Clathrin-coated vesicles contain an ATP-dependent proton pump.
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 microM) and vanadate (10 microM) and the mitochondrial ATPase inhibitors oligomycin (10 microgram/ml) and aurovertin (1 microgram/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.
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D002966:Clathrin; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D011992:Endosomes / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins; D008744:Methylamines / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D006180:Proton-Translocating ATPases; D014634:Valinomycin / Q000494:pharmacology
D002966:Clathrin; D008565:Membrane Proteins; D008744:Methylamines; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C027451:methylamine; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D008274:Magnesium; D011188:Potassium