Photoaffinity labeling of wild-type and mutant forms of the yeast V-ATPase A subunit by 2-azido-[(32)P]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 subunit (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 residues to the adenine ring, the yeast V-ATPase containing wild-type and mutant forms of the A subunit was reacted with 2-azido-[(32)P]ADP, a photoaffinity analog that stably modifies tyrosine but not phenylalanine residues. Mutant forms of the A subunit were constructed in which the two endogenous tyrosine residues were replaced with phenylalanine and in which a single tyrosine was introduced at each of the four positions. Strong ATP-protectable labeling of the A subunit was observed for the wild-type and the mutant containing tyrosine at 532, significant ATP-protectable labeling was observed for the mutants containing tyrosine at positions 452 and 538, and only very weak labeling was observed for the mutants containing tyrosine at 535 or in which all four residues were phenylalanine. These results suggest that Tyr(532) 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 at Phe(452), Tyr(532), Tyr(535), and Glu(286) on dissociation of the peripheral V(1) and integral V(0) domains both in vivo and in vitro were examined. The results suggest that in vivo dissociation requires catalytic activity while in vitro dissociation requires nucleotide binding to the catalytic site.
MacLeod
K J
KJ
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Vasilyeva
E
E
Merdek
K
K
Vogel
P D
PD
Forgac
M
M
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009154:Mutation; D010761:Phosphorus Radioisotopes; D011193:Potassium Iodide / Q000494:pharmacology; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays; D025262:Vacuolar Proton-Translocating ATPases; D015003:Yeasts / Q000201:enzymology
D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; D011193:Potassium Iodide; D014443:Tyrosine; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases