Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex.
Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.
Xie
X S
XS
Stone
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D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure; D002417:Cattle; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*
D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide