Abstract
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 V(1) subunit (vma2Delta), V(o) subunit, or either of the two V(o) a subunit isoforms are acutely sensitive to H(2)O(2) and more sensitive to menadione and diamide than wild-type cells. The vma2Delta 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. vma2Delta 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 H(2)O(2). Microarray analysis comparing wild-type and vma2Delta mutant cells grown at pH 5, permissive conditions for the vma2Delta 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.
Highlights
Both prokaryotic and eukaryotic cells have multiple mechanisms for preventing and counteracting the effects of oxidative stress [1]
We deliberately chose to assess sensitivity to oxidative stress in strains containing deletions in a V1 sector subunit and a Vo sector subunit that are both found in all yeast V-ATPases as well as the two Vo a subunit isoforms, each found in a subset of V-ATPases
We found that all were sensitive, suggesting that sensitivity is a general feature of loss of V-ATPase activity
Summary
Materials and Strains—Hydrogen peroxide was obtained from Acros. Menadione and diamide were obtained from Sigma. Halo assays for sensitivity to various oxidants were performed as described [22], except that the indicated concentrations of oxidant were added to the filter paper disk after the disk was placed on an overlay of cells grown to log phase in liquid culture, mixed with an equal volume of 1% agar, and layered onto a YEPD plate buffered to either pH 7 or pH 5. Liquid cultures of BY4741 and BY4741 vma2⌬ strains and the corresponding rho0 cells were grown overnight in SC, and DHR was added to a final concentration of 5 g/ml. Microarray and Northern Analyses—Total yeast RNA was isolated from wild-type and vma2⌬ mutant yeast cells grown to early log phase in YEPD, pH 5, as described [26]. Blots were hybridized with the probes as described [28] and washed and analyzed on an Amersham Biosciences Typhoon PhosphorImager
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