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J. Biol. Chem., Vol. 282, Issue 10, 7125-7136, March 9, 2007

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Loss of Vacuolar Proton-translocating ATPase Activity in Yeast Results in Chronic Oxidative Stress^*

Elena Milgrom¹, Heba Diab¹, Frank Middleton, and Patricia M. Kane²

From the Departments of Biochemistry and Molecular Biology and Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, New York 13210

Yeast mutants lacking vacuolar proton-translocating ATPase (V-ATPase) subunits (vma mutants) were sensitive to several different oxidants in a recent genomic screen (Thorpe, G. W., Fong, C. S., Alic, N., Higgins, V. J., and Dawes, I. W. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 6564–6569). We confirmed that mutants lacking a V₁ subunit (vma2), V_o subunit, or either of the two V_o a subunit isoforms are acutely sensitive to H₂O₂ and more sensitive to menadione and diamide than wild-type cells. The vma2 mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2 mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H₂O₂. Microarray analysis comparing wild-type and vma2 mutant cells grown at pH 5, permissive conditions for the vma2 mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.

Received for publication, August 30, 2006 , and in revised form, December 13, 2006.

^* This work was supported by National Institutes of Health Grant R01-GM50322 (to P. M. K.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Tables 1 and 2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210. Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm{at}upstate.edu.

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