Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex

X. S. Xie, D. K. Stone

Research output: Contribution to journalArticle

88 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2492-2495
Number of pages4
JournalJournal of Biological Chemistry
Volume261
Issue number6
StatePublished - Dec 1 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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