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J. Biol. Chem., Vol. 280, Issue 49, 40481-40488, December 9, 2005

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Subunit a of the Yeast V-ATPase Participates in Binding of Bafilomycin^*

From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

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 V₀ 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 Arg⁷³⁵ 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.

Received for publication, August 17, 2005 , and in revised form, October 6, 2005.

^* This work was supported by National Institutes of Health Grant GM34478 (to M. F.) and a postdoctoral fellowship from the Northeast Affiliate of the American Heart Association (to T. I.). E. coli strains were provided through National Institutes of Health Grant DK34928. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail: michael.forgac{at}tufts.edu.

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