Increased cellular levels of reactive oxygen species are known to arise during exposure of organisms to elevated metal concentrations, but the consequences for cells in the context of metal toxicity are poorly characterized. Using two-dimensional gel electrophoresis, combined with immunodetection of protein carbonyls, we report here that exposure of the yeast Saccharomyces cerevisiae to copper causes a marked increase in cellular protein carbonyl levels, indicative of oxidative protein damage. The response was time dependent, with total-protein oxidation peaking approximately 15 min after the onset of copper treatment. Moreover, this oxidative damage was not evenly distributed among the expressed proteins of the cell. Rather, in a similar manner to peroxide-induced oxidative stress, copperdependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoformspeci¢c and, in most cases, was also associated with a decline in the proteins' relative abundance. Our results are consistent with a model in which copper-induced oxidative stress disables the £ow of carbon through the preferred glycolytic pathway, and promotes the production of glucose-equivalents within the pentose phosphate pathway. Such re-routing of the metabolic £ux may serve as a rapid-response mechanism to help cells counter the damaging e¡ects of copper-induced oxidative stress. ß

Saccharomyces cerevisiae expresses multiple phospholipid hydroperoxide glutathione peroxidase (PHGPx)-like proteins in the absence of a classical glutathione peroxidase (cGPx), providing a unique system for dissecting the roles of these enzymes in vivo. The Gpx3 (Orp1/PHGpx3) protein transduces the hydroperoxide signal to the transcription factor Yap1, a function that could account for most GPX-dependent phenotypes. To test this hypothesis and ascertain what functions of Gpx3 can be shared by cGPx-like enzymes, we constructed a novel cGPx-like yeast enzyme, cGpx3. We confirmed that the "gap" sequences conserved among cGPxs but absent from aligned PHGPx sequences are the principal cause of the structural and functional differences of these enzymes. Peroxidase activity against a cGPx substrate was high in the cGpx3 construct, which was multimeric and had a peroxidase catalytic mechanism distinct from Gpx3; but cGpx3 was defective for phospholipid hydroperoxidase and signaling activities. cGpx3 did not complement the sensitivity to lipid peroxidation of a gpxDelta mutant, and the resistance to lipid peroxidation conferred by Gpx3 was independent of Yap1, establishing a functional role for Gpx3 phospholipid hydroperoxidase activity. Using the comparison between cGpx3 and Gpx3 in conjunction with other constructs to probe lipid peroxidation as a toxicity mechanism, we also ascertained that lipid peroxidation-dependent processes are a principal cause of cellular cadmium toxicity. The results demonstrate that phospholipid hydroperoxidase and Yap1-mediated signaling activities of Gpx3 have independent functional roles, although both functions depend on the absence of cGPx-like subunit interaction sites, and the results resolve more clearly the potential drivers of the differential selective evolution of GPx-like enzymes.

Oxidative damage in microbial cells occurs during exposure to the toxic metal chromium, but it is not certain whether such oxidation accounts for the toxicity of Cr. Here, a Saccharomyces cerevisiae sod1D mutant (defective for the Cu,Zn-superoxide dismutase) was found to be hypersensitive to Cr(VI) toxicity under aerobic conditions, but this phenotype was suppressed under anaerobic conditions. Studies with cells expressing a Sod1p variant (Sod1 H46C ) showed that the superoxide dismutase activity rather than the metal-binding function of Sod1p was required for Cr resistance. To help identify the macromolecular target(s) of Cr-dependent oxidative damage, cells deficient for the reduction of phospholipid hydroperoxides (gpx3D and gpx1D/gpx2D/gpx3D) and for the repair of DNA oxidation (ogg1D and rad30D/ogg1D) were tested, but were found not to be Cr-sensitive. In contrast, S. cerevisiae msraD (mxr1D) and msrbD (ycl033cD) mutants defective for peptide methionine sulfoxide reductase (MSR) activity exhibited a Cr sensitivity phenotype, and cells overexpressing these enzymes were Cr-resistant. Overexpression of MSRs also suppressed the Cr sensitivity of sod1D cells. The inference that protein oxidation is a primary mechanism of Cr toxicity was corroborated by an observed~20-fold increase in the cellular levels of protein carbonyls within 30 min of Cr exposure. Carbonylation was not distributed evenly among the expressed proteins of the cells; certain glycolytic enzymes and heat-shock proteins were specifically targeted by Cr-dependent oxidative damage. This study establishes an oxidative mode of Cr toxicity in S. cerevisiae, which primarily involves oxidative damage to cellular proteins.

Methionine residues and iron-sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of micro-organisms. Here, we show that methionine redox modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxrD double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild-type reproduced the Cu-resistance phenotype, and FeS-cluster functions were found to be defective in the mxrD mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. 55 Fe-labelling studies showed that FeS clusters are turned over more rapidly in the mxrD mutant than the wild-type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes in helping to protect the essential function of FeS clusters in aerobic settings. INTRODUCTIONReactive oxygen species (ROS) are generated continuously through the process of respiration in all aerobic organisms, and may cause the oxidative deterioration of lipid, DNA and protein function in cells (Temple et al., 2005). ROS damage is often exacerbated during stress. To counter this, cells have evolved a range of enzymic and non-enzymic antioxidant defence mechanisms. The majority of these serve a preventative role in scavenging ROS before they exert damage. Others repair incurred oxidative damage, including phospholipid hydroperoxide glutathione peroxidases (lipid peroxidation) (Avery et al., 2004) and 8-oxoG DNA glycosylases (DNA oxidation) (Boiteux & Radicella, 1999). The principal enzymic mechanism for reversing protein oxidation acts on the oxidation product of just one amino acid residue, methionine. This specificity for Met reflects the fact that Met is particularly susceptible to oxidation compared with other amino acids. Furthermore, Met oxidation has been linked to Alzheimer's (Schoneich, 2005) and Parkinson's (Wassef et al., 2007) diseases. Methionine sulphoxide reductases (MSRs) are conserved across nearly all organisms from bacteria to humans (Kryukov et al., 2002), and have been the focus of considerable attention in recent years. Two MSR activities have been characterized in the yeast Saccharomyces cerevisiae: MsrA (encoded by MXR1) reduces the S stereoisomer of methionine sulphoxide (MetO), while MsrB (e...