Subunit a of the yeast V-ATPase participates in binding of bafilomycin

Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H(+))-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V(0) domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed K(i) values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nm). These included E721K (K(i) 0.38 +/- 0.03 nm), L724A (0.40 +/- 0.02 nm), and N725F (0.54 +/- 0.06 nm). Only the N725F mutation displayed a K(i) for concanamycin (0.84 +/- 0.04 nm) that was slightly higher than wild-type (0.60 +/- 0.07 nm). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.