ISOLATION OF A PROTEIN ACTIVATOR OF THE CLATHRIN-COATED VESICLE PROTON PUMP

An activator of the clathrin-coated vesicle proton translocating ATPas e has been purified 1600-fold from bovine brain. The activator, which requires detergent (polyoxyethylene 9-lauryl ether) for release from c lathrin-coated vesicles, is heat-stable, trypsin-sensitive, and has an apparent molecular mass of about 6 kDa as determined by high performa nce liquid chromotography. The activator stimulates the purified H+-AT Pase of coated vesicles over 50-fold under acidic conditions. Similarl y, the activator stimulates proton pumping catalyzed by the reconstitu ted proton pump. Importantly, this stimulation of proton pumping is ob served only when the activator is reconstituted into the interior of t he proteoliposomes. Moreover, the activator protein is demonstrated to protect, and co-sediment with, purified proton pump during glycerol g radient centrifugation performed in the presence of ATP. These observa tions support the notion that this activator serves to determine the p H set point of acidic endomembranes through interactions with the tran smembranous sectors of the proton pump.