Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex.
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 mumol 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.
D000818:Animals; D001436:Bacteriorhodopsins / Q000494:pharmacology; D002234:Carbodiimides / Q000494:pharmacology*; D002417:Cattle; D002966:Clathrin / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology*; D008928:Mitochondria / Q000201:enzymology; D010455:Peptides / Q000302:isolation & purification*; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014638:Vanadates / Q000494:pharmacology
D002234:Carbodiimides; D002966:Clathrin; D010455:Peptides; D014638:Vanadates; D001436:Bacteriorhodopsins; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases