Photoaffinity labeling of wild-type and mutant forms of the yeast V-ATPaseA subunit by 2-azido-[P-32]ADP

Molecular modeling studies have previously suggested the possible presence of four aromatic residues (Phe(452), Tyr(532), Tyr(535), and Phe(538)) near the adenine binding pocket of the catalytic site on the yeast V-ATPase A s ubunit (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). To test the proximity of these aromatic resi dues to the adenine ring, the yeast V-ATPase containing wild-type and mutan t forms of the A subunit was reacted with 2-azido-[P-32]ADP, a photoaffinit y analog that stably modifies tyrosine but not phenylalanine residues. Muta nt forms of the A subunit were constructed in which the two endogenous tyro sine residues were replaced with phenylalanine and in which a single tyrosi ne was introduced at each of the four positions, Strong ATP-protectable lab eling of the A subunit was observed for the wild-type and the mutant contai ning tyrosine at 532, significant ATP-protectable labeling was observed for the mutants containing tyrosine at positions 452 and 538, and only very we ak labeling was observed for the mutants containing tyrosine at 535 or in w hich all four residues were phenylalanine. These results suggest that Tyr53 2 and possibly Phe(452) and Tyr(538) are in close proximity to the adenine ring of ATP bound to the A subunit, In addition, the effects of mutations a t Phe(452), Tyr(532), Tyr(535), and Glu(286) on dissociation of the periphe ral V-1 and integral V-0 domains both in vivo and in vitro were examined. T he results suggest that in vivo dissociation requires catalytic activity wh ile in vitro dissociation requires nucleotide binding to the catalytic site .