THE COATED VESICLE VACUOLAR (H-ATPASE ASSOCIATES WITH AND IS PHOSPHORYLATED BY THE 50-KDA POLYPEPTIDE OF THE CLATHRIN ASSEMBLY PROTEIN AP-2())

We have previously noted a 50-kDa polypeptide (p50) co-purifying with preparations of the bovine brain clathrin-coated vesicle vacuolar (H+) -ATPase (V-ATPase) (Zhang, J., Myers, M., and Forgac, M. (1992) J. Bio l. Chem. 267,9773-9778). We show that p50 is also immunoprecipitated w ith the V-ATPase, further suggesting its specific association with the proton pump. To determine the identity of this 50-kDa polypeptide and the stoichiometry of its association with the V-ATPase, we performed N-terminal amino acid sequencing and quantitative amino acid analysis of the gel-purified protein. These results revealed the unknown polype ptide to be the 50-kDa subunit of the clathrin assembly protein AP-2 ( AP50); we estimate the stoichiometry of association is one AP50 per V- ATPase complex. AP50 is an N-ethylmaleimide (NEM)-inhibitable autokina se and incubation of purified V-ATPase with [gamma-P-32]ATP resulted i n the NEM-sensitive phosphorylation of AP50 and the B subunit of the V -ATPase. The same phosphorylation pattern is seen if the labeling reac tion is done with intact clathrin-coated vesicles and the V-ATPase sub sequently immunoprecipitated from the solubilized vesicles. This repre sents the first report of phosphorylation of one of the V-ATPase subun its. The functional significance of this phosphorylation for regulatio n or targeting of the V-ATPase in vivo remains to be determined.