Inhibition and Labeling of the Coated Vesicle V-ATPase by 2-Azido-32PATP

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-32PATP have been carried out. We have observed that 2-azido-32PATP is hydrolyzed by the V-ATPase at a rate (at 1 mM) approximately 4-fold lower than observed for ATP, indicating that 2-azido-32PATP is a good substrate for the V-ATPase. Irradiation of the V-ATPase in the presence of 0.5 mM 2-azido-32PATP 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-32PATP. The time course for inactivation most closely correlates with labeling of the A subunit. Measurement of the stoichiometry of 2-azido-32PATP 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-32PATP/mol V-ATPase complex. 2-Azido-32PATP labeling indicates that the V-ATPase possesses both rapidly (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-32PATP-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.(ABSTRACT TRUNCATED AT 400 WORDS)