Inhibition and labeling of the coated vesicle V-ATPase by 2-azido-[32P]ATP

Abstract

Previous studies have indicated that the 73-kDa A subunit of the coated vesicle V-ATPase possesses a nucleotide-binding site essential for activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638) and have identified a cysteine residue (Cys254) whose modification leads to complete loss of activity (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). To further characterize the structure of the nucleotide-binding sites of the V-ATPase, labeling studies using the photoactivated analog 2-azido-[32P]ATP have been carried out. We have observed that 2-azido-[32P]ATP is hydrolyzed by the V-ATPase at a rate (at 1 mM) approximately 4-fold lower than observed for ATP, indicating that 2-azido-[32P]ATP is a good substrate for the V-ATPase. Irradiation of the V-ATPase in the presence of 0.5 mM 2-azido-[32P]ATP leads to inactivation of V-ATPase activity with a t1/2 of 3-5 min. The 73-kDa A subunit, the 58-kDa B subunit, and the 50-kDa subunit of the AP-2 adaptin complex (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186) are all labeled in an ATP-protectable manner on irradiation of the purified V-ATPase with 2-azido-[32P]ATP. The time course for inactivation most closely correlates with labeling of the A subunit. Measurement of the stoichiometry of 2-azido-[32P]ATP incorporation into the A subunit as a function of inactivation indicates that complete loss of activity is obtained on incorporation of 1.2 mol of 2-azido-[32P]ATP/mol V-ATPase complex. 2-Azido-[32P]ATP labeling indicates that the V-ATPase possesses both rapidly (t1/2 < 2 min) and slowly (t1/2 > 2 min) exchangeable nucleotide-binding sites. The A subunit is labeled upon modification of both rapidly and slowly exchangeable sites whereas the B subunit is labeled upon modification of only rapidly exchangeable sites. Inhibition of V-ATPase activity correlates with labeling of the rapidly exchangeable sites. Amino acid sequence analysis of peptides derived from the 2-azido-[32P]ATP-labeled A subunit indicates labeling of two peptides: a 12-kDa fragment which begins at residue 511 and contains Cys532 and a 3-kDa fragment which begins at residue 233 and contains the glycine-rich loop and Cys254. Only the 12-kDa fragment is labeled upon modification of the rapidly exchangeable sites. A 12-kDa V8 peptide, which begins at residue 375 of the B subunit is also labeled by 2-azido-[32P] ATP under conditions which modify the rapidly exchangeable sites. These results provide further information concerning the nucleotidebinding sites on the V-ATPase A and B subunits. The differences in nucleotide-binding sites between F-and V-ATPases are also discussed.