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

S. Z. Sun, X. S. Xie, D. K. Stone

Research output: Contribution to journalArticle

63 Scopus citations

Abstract

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 μmol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.

Original languageEnglish (US)
Pages (from-to)14790-14794
Number of pages5
JournalJournal of Biological Chemistry
Volume262
Issue number30
StatePublished - Jan 1 1987

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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