Aerobic organisms have to maintain a reduced cellular redox environment in the face of the prooxidative conditions of aerobic life. The incomplete reduction of oxygen to water during respiration leads to the formation of redox-active oxygen intermediates such as the superoxide anion radical (O 2 . ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (for review see Refs.

Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium.Heavy metals represent major environmental hazards to human health. In particular, cadmium is very toxic and probably carcinogenic at low concentrations. However, the biological effects of this metal and the mechanism of its toxicity are not yet clearly understood. It has been proposed that Cd 2ϩ ions might displace Zn 2ϩ and Fe 2ϩ in proteins (1), resulting in their inactivation and in the release of free iron, which might generate highly reactive hydroxyl radicals (OH ⅐ ) (2). In support of this hypothesis, a major effect of cadmium is oxidative stress (3), particularly lipid peroxidation (1). However, it is not known whether these effects are responsible for the extreme toxicity of the metal.Living organisms use several mechanisms to counter cadmium toxicity. In bacteria, efflux pumps are able to export toxic ions outside the cell (4). In higher eukaryotes, Cd 2ϩ is sequestered by metallothioneins through their high cysteine content (5). Cadmium can also be detoxified by chelation to GSH or to phytochelatin, a glutathione polymer of general structure (␥-Glu-Cys) n -Gly synthesized from GSH in plants and in the yeast Schizosaccharomyces pombe. Cd 2ϩ -phytochelatin and Cd 2ϩ ⅐ (GSH) 2 complexes are transported into the vacuole by ATPbinding cassette transporters (6 -8).Yap1p and Skn7p are yeast transcription factors that regulate the adaptive response to oxidative stress (9 -11). Strains lacking either transcription factor are sensitive to H 2 O 2 and are defective in the induction by H 2 O 2 of several enzymes with antioxidant properties (9). Yap1p is also important in cadmium tolerance because yap1-deleted strains are very sensitive to cadmium, and strains overexpressing YAP1 are hyper-resistant to this toxic metal (12). The contribution of Skn7p to the cadmium response is more complex, because skn7-deleted strains are hyper-resistant to cadmium (9)...

To isolate new antioxidant genes, we have searched for activities that would rescue the tert-butyl hydroperoxide (t-BOOH)-hypersensitive phenotype of a Saccharomyces cerevisiae strain deleted for the gene encoding the oxidative stress response regulator Skn7. We report the characterization of AHP1, which encodes a 19-kDa protein similar to the AhpC/TSA protein family within a small region encompassing Cys-62 of Ahp1p and the highly conserved N-terminal catalytic AhpC/TSA cysteine. Ahp1p contains a peroxisomal sorting signal, suggesting a peroxisomal localization. AHP1 exerts strong antioxidant protective functions, as demonstrated both by gene overexpression and deletion analyses, and is inducible by peroxides in an Yap1-and Skn7-dependent manner. Similar to yeast Tsa1p, Ahp1p forms a disulfidelinked homodimer upon oxidation and in vivo requires the presence of the thioredoxin system but not of glutathione to perform its antioxidant protective function. Furthermore, in contrast to Tsa1p, which is specific for H 2 O 2 , Ahp1p is specific for organic peroxides. Therefore, with respect to substrate specificity, Ahp1p differs from Tsa1p and is similar to prokaryotic alkyl hydroperoxide reductase AhpC. These data suggest that Ahp1p is a yeast orthologue of prokaryotic AhpC and justifies its name of yeast alkyl hydroperoxide reductase.The incomplete reduction of molecular oxygen during respiration and the lipid metabolism in peroxisomes leads to the formation of reactive oxygen species (ROS) 1 (1). ROS are potent oxidants and can damage all cellular constituents (2). They cause DNA base modifications and strand breaks and are therefore mutagenic. They can damage proteins and inactivate enzymes. Oxidation of membrane lipids can initiate free radical chain reactions, which alter cellular membranes and give rise to other very toxic reactive species such as lipid hydroperoxides, alkoxyl and peroxyl radicals, lipid epoxides, and aldehydes. To protect against the toxicity of ROS, aerobic organisms use an array of defense mechanisms (1, 3, 4). Among these, cytosolic and mitochondrial superoxide dismutases eliminate the O 2 . radical, cytosolic and peroxisomal catalases remove H 2 O 2 , and glutathione peroxidases reduce both H 2 O 2 and alkyl hydroperoxides. The AhpC/TSA family, also referred to as peroxiredoxin, is a large family of newly discovered peroxidases that are highly conserved from prokaryotes to eukaryotes (5) and act to reduce hydroperoxides with electrons donated by NADPH via thioredoxin or other thiol-containing intermediates (6 -8). Antioxidant defense mechanisms are induced as part of global cellular adaptive responses to oxidative stress (9 -12). In Saccharomyces cerevisiae, the cellular response to hydroperoxides is associated with the induction of a very large stimulon of at least 115 proteins (13), which involves, at least in part, the transcriptional regulators Yap1 (14, 15) and Skn7 (16,17). Strains inactivated for either one of these regulators are hypersensitive to killing by H 2 O 2 and by several o...

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Abstract. A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. CellRes., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaC1 soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus.Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS pop yacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits.Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections.

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In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of ⌬gsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of ⌬gsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that ␥-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a ⌬gsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions. Glutathione (GSH) is a broadly conserved tripeptide with a highly reactive thiol and a very low redox potential of about Ϫ240 to Ϫ250 mV. Its elevated concentration, up to 10 mM, and the fact that its reduced state is efficiently maintained by NADPH-dependent glutathione reductase (reviewed in Refs. 1-3) confers to this small molecule the properties of a cellular redox buffer. As a redox buffer, GSH is thought to be a major determinant in maintaining a reducing cellular thiol-disulfide balance. GSH is also important as an electron donor for several enzymes that have a reducing step in their catalytic cycle, such as ribonucleotide reductase (4). In these situations, electrons from GSH are generally transduced to their substrates through glutaredoxins. GSH may protect protein sulfhydryls from irreversible oxidation by glutathionylation (5, 6). It also participates in the detoxification of peroxides, either directly or indirectly as a cofactor of glutathione peroxidase (1,7,8) and of several chemicals such as cadmium (9, 10).In Saccharomyces cerevisiae and in probably all other GSHcontaining organisms, GSH is synthesized in two steps beginning with the action of ␥-glutamyl cysteine synthetase (GSH1) (11, 12), which catalyzes the condensation of glutamic acid to cysteine, in the rate-limiting step of this biosynthetic pathway. The product of this reaction is ␥-glutamylcysteine, which is combined with glycine through the action of glutathione synthase (GSH2) (13) to form GSH. Null mutations in GSH1 result in GSH auxotrophy (14 -16), demonstrating that GSH is essential in yeast. In mammals, GSH is also essential, as demonstrated by the embryonic lethality resulting from disruption of ␥-glutamyl cysteine synthetase ...

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