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. Biol. Chem. 267, 9773-9778). We show that p50 is also immunoprecipitated with 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 polypeptide 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 autokinase and incubation of purified V-ATPase with [gamma-32P]ATP resulted in the NEM-sensitive phosphorylation of AP50 and the B subunit of the V-ATPase. The same phosphorylation pattern is seen if the labeling reaction is done with intact clathrin-coated vesicles and the V-ATPase subsequently immunoprecipitated from the solubilized vesicles. This represents the first report of phosphorylation of one of the V-ATPase subunits. The functional significance of this phosphorylation for regulation or targeting of the V-ATPase in vivo remains to be determined.
Myers
M
M
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Forgac
M
M
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D046911:Macromolecular Substances; D010750:Phosphoproteins; C438885:adaptor protein complex 2, mu 1 subunit; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide