SummaryGlutaredoxins and thioredoxins are highly conserved, small, heat-stable oxidoreductases. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2), and we have used multiple mutants to determine their roles in mediating resistance to oxidative stress caused by hydroperoxides. Our data indicate that TRX2 plays the predominant role, as mutants lacking TRX2 are hypersensitive, and mutants containing TRX2 are resistant to these oxidants. However, the requirement for TRX2 is only apparent during stationary phase growth, and we present three lines of evidence that the thioredoxin isoenzymes actually have redundant activities as antioxidants. First, the trx1 and trx2 mutants show wild-type resistance to hydroperoxide during exponential phase growth; secondly, overexpression of either TRX1 or TRX2 leads to increased resistance to hydroperoxides; and, thirdly, both Trx1 and Trx2 are equally able to act as cofactors for the thioredoxin peroxidase, Tsa1. The antioxidant activity of thioredoxins is required for both the survival of yeast cells as well as protection against oxidative stress during stationary phase growth, and correlates with an increase in the expression of both TRX1 and TRX2. We show that the requirement for thioredoxins during this growth phase is dependent on their activity as cofactors for the antioxidant enzyme Tsa1, and for regulation of the redox state and proteinbound levels of the low-molecular-weight antioxidant glutathione. IntroductionMany reports have highlighted the key role played by sul- dispensable during normal growth conditions (Luikenhuis et al., 1997). Unlike the thioredoxins, however, loss of glutaredoxins does not affect the cell cycle, nor does it result in any detectable aberrant growth phenotype.As in other species, yeast thioredoxins and glutaredoxins are active as antioxidants and play key roles in protection against oxidative stress induced by various reactive oxygen species (ROS). TRX2 was originally identified as a target gene of the yAP-1 transcriptional activator protein, which regulates the expression of a large number of antioxidants in response to oxidative stress (Kuge and Jones, 1994;Toone and Jones, 1999). Furthermore, TRX2 was shown to be essential for yAP-1-mediated resistance to hydroperoxides (Kuge and Jones, 1994). Thioredoxins are also required for the detoxification of ROS through their reactivity with thioredoxin peroxidases. A large number of thioredoxin peroxidase isoforms have been identified in yeast, including cytoplasmic (Tsa1, cTpx2/YDR453c, Tsa2/Ahp1/ YLR109), mitochondrial (mTpx/YBL064c) and nuclear (nTpx/YIL010w) (Chae et al., 1993;Jeong et al., 1999;Verdoucq et al., 1999;Park et al., 2000;Pedrajas et al., 2000). Enzyme analysis has confirmed that all the gene products display thioredoxin peroxidase activity, but their exact intracellular roles and targets remain unclear (Park et al., 2000). Strains deleted for both GRX1 and GRX2 lack heat-stable oxidoreductase activi...

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.All aerobic organisms are exposed to reactive oxygen species (ROS), 1 such as H 2 O 2 , the superoxide anion, and the hydroxyl radical during the course of normal aerobic metabolism or following exposure to radical-generating compounds. These ROS can cause wide-ranging damage to cells, and an oxidative stress is said to occur when the cellular survival mechanisms are unable to cope with the ROS or the damage they cause (1). Oxidative damage is associated with various diseases such as cancer, vascular, and neurodegenerative disorders, as well as with aging processes (2-4). To protect against damage, cells contain a number of defense mechanisms including enzymes, such as catalase, superoxide dismutase, glutathione peroxidase, and low molecular weight antioxidants such as glutathione (GSH) and vitamins C and E (5, 6). Recent studies have highlighted the key role played by sulfhydryl groups (-SH) in the response to oxidative stress, and in particular, the roles of the GSH/glutaredoxin and thioredoxin systems, which maintain the redox homeostasis of the cell (7-10). In this present study, we examine the role of yeast glutaredoxins in protection against hydroperoxides.Glutaredoxins are small heat-stable oxidoreductases, first discovered in Escherichia coli as GSH-dependent hydrogen donors for ribonucleotide reductase (11). They form part of the glutaredoxin system, comprising NADPH, GSH, and glutathione reductase, which transfers electrons from NADPH to glutaredoxins via GSH (12). The yeast Saccharomyces cerevisiae contains two glutaredoxins, designated Grx1 and Grx2, which share 40 -52% identity and 61-76% similarity with those...