Inhibition of the coated vesicle proton pump and labeling of a 17,000-dalton polypeptide by N,N'-dicyclohexylcarbodiimide.
N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits 100% of proton transport and 80-85% of (Mg2+)-ATPase activity in clathrin-coated vesicles. Half-maximum inhibition of proton transport is observed at 10 microM DCCD after 30 min. Although treatment of the coated vesicle (H+)-ATPase with DCCD has no effect on ATP hydrolysis in the detergent-solubilized state, sensitivity of proton transport and ATPase activity to DCCD is restored following reconstitution into phospholipid vesicles. In addition, treatment of the detergent-solubilized enzyme with DCCD followed by reconstitution gives a preparation that is blocked in both proton transport and ATP hydrolysis. These results suggest that although the coated vesicle (H+)-ATPase can react with DCCD in either a membrane-bound or detergent-solubilized state, inhibition of ATPase activity is only manifested when the pump is present in sealed membrane vesicles. To identify the subunit responsible for inhibition of the coated vesicle (H+)-ATPase by DCCD, we have labeled the partially purified enzyme with [14C]DCCD. A single polypeptide of molecular weight 17,000 is labeled. The extremely hydrophobic nature of this polypeptide is indicated by its extraction with chloroform:methanol. The 17,000-dalton protein can be labeled to a maximum stoichiometry of 0.99 mol of DCCD/mol of protein with 100% inhibition of proton transport occurring at a stoichiometry of 0.15-0.20 mol of DCCD/mol of protein. Amino acid analysis of the chloroform:methanol extracted 17,000-dalton polypeptide reveals a high percentage of nonpolar amino acids. The similarity in properties of this protein and the DCCD-binding subunit of the coupling factor (H+)-ATPases suggests that the 17,000-dalton polypeptide may function as part of a proton channel in the coated vesicle proton pump.
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors; D002234:Carbodiimides / Q000494:pharmacology*; D002417:Cattle; D002966:Clathrin; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology*; D011992:Endosomes / Q000201:enzymology*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D011522:Protons
D002234:Carbodiimides; D002966:Clathrin; D007473:Ion Channels; D008081:Liposomes; D011522:Protons; D004024:Dicyclohexylcarbodiimide; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases