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J. Biol. Chem., Vol. 279, Issue 32, 33131-33138, August 6, 2004

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The Bafilomycin/Concanamycin Binding Site in Subunit c of the V-ATPases from Neurospora crassa and Saccharomyces cerevisiae^*

Emma Jean Bowman, Laurie A. Graham, Tom H. Stevens, and Barry J. Bowman¶

From the Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064 and the Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

The vacuolar H⁺-ATPase is inhibited with high specificity and potency by bafilomycin and concanamycin, macrolide antibiotics with similar structures. We previously reported that mutation at three residues in subunit c of the vacuolar ATPase from Neurospora crassa conferred strong resistance to bafilomycin but little or no resistance to concanamycin (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). We have identified additional mutated sites in subunit c that confer resistance to bafilomycin. Furthermore, by subjecting a resistant mutant to a second round of mutation we isolated strains with increased resistance to both bafilomycin and concanamycin. In all of these strains the second mutation is also in subunit c, suggesting it forms at least part of the concanamycin binding site. Site-directed mutagenesis of the gene encoding subunit c in Saccharomyces cerevisiae showed that single mutations in each of the residues identified in one of the double mutants of N. crassa conferred resistance to both bafilomycin and concanamycin. Mutations at the corresponding sites in the VMA11 and VMA16 genes of S. cerevisiae, which encode the c' and c'' subunits, did not confer resistance to the drugs. In all, nine residues of subunit c have been implicated in drug binding. The positions of these residues support a model in which the drug binding site is a pocket formed by helices 1, 2, and 4. We hypothesize that the drugs inhibit by preventing the rotation of the c subunits.

Received for publication, April 26, 2004 , and in revised form, May 27, 2004.

^* This work was supported in part by National Institutes of Health Grants GM38006 (to T. H. S.) and GM58903 (to B. J. B.). 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.

^¶ To whom correspondence should be addressed. Tel.: 831-459-2245; Fax: 831-459-3139; E-mail: bowman{at}biology.ucsc.edu.

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