Abstract
Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest. Here, we show that the Yap1 and Met4 transcription factors regulate the expression of gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion. Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered. Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response. The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1. In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion. Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation. These results indicate that the Yap1-dependent activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependent mechanism.
Highlights
The ability to regulate biosynthetic pathways is a fundamental aspect of adaptation to life in a changing environment
In this paper we have investigated the mechanisms regulating the biosynthesis of the key antioxidant, GSH
Given that thioredoxins are required for Yap1 deactivation [22], we propose that thioredoxin oxidation resulting from GSH depletion is responsible for the activation of Yap1 and leads to the increased biosynthesis of GSH
Summary
Strain CY813, which is deleted for CBF1, was made by back-crossing CY4 with EUROSCARF strain Y16858 (MAT␣ his3D1 leu2D0 lys2D0 ura3D0 cbf1::kanMX4) These strains were used to construct the gsh yap, gsh cbf, met cbf, yap cbf, gsh met cbf, and gsh yap cbf mutants using standard yeast genetic methods. Error bars denote S.E. Northern Blot Analysis—Yeast cells were grown to mid-exponential phase (A600 ϭ 0.6 – 0.7) in minimal media in the presence of 1 mM methionine or 1 mM GSH where appropriate. Microarray Hybridizations and Data Analysis—Yeast cells were grown in triplicate to mid-exponential phase in minimal SD media. Preparation of RNA, probes, and hybridization to whole yeast genome microarrays (YG-S98, Affymetrix) was performed as described on the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) web site (www.cogeme.man.ac.uk). The identification of prospective regulatory sequences within promoters was performed by RSAT (Regulatory Sequence Analysis Tools, rsat.ulb.ac.be/rsat)
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