The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1⌬, gpx2⌬, and gpx3⌬ deletion mutants; PHGPx activity was almost absent in a gpx1⌬/ gpx2⌬/gpx3⌬ triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1⌬/gpx2⌬/ gpx3⌬ mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H 2 O 2 . Unlike H 2 O 2 toxicity, delayed toxicity of 18:3 toward gpx1⌬/gpx2⌬/ gpx3⌬ cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with ␣-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.

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...

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.

Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light. An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized. In the present study we present data from a detailed substrate specificity trial. We have determined that the substrate range of Uve1p is much greater than was originally believed. We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule. We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites. This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described. This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.

SummaryPhenotypic heterogeneity describes non-genetic variation that exists between individual cells within isogenic populations. The basis for such heterogeneity is not well understood, but it is evident in a wide range of cellular functions and phenotypes and may be fundamental to the fitness of microorganisms. Here we use a suite of novel assays applied to yeast, to provide an explanation for the classic example of heterogeneous resistance to stress (copper). Cell cycle stage and replicative cell age, but not mitochondrial content, were found to be principal parameters underpinning differential Cu resistance: cell cyclesynchronized cells had relatively uniform Cu resistances, and replicative cell-age profiles differed markedly in sorted Cu-resistant and Cu-sensitive subpopulations. From a range of potential Cu-sensitive mutants, cup1 D cells lacking Cu-metallothionein, and particularly sod1 D cells lacking Cu, Zn-superoxide dismutase, exhibited diminished heterogeneity. Furthermore, age-dependent Cu resistance was largely abolished in cup1 D and sod1 D cells, whereas cell cycle-dependent Cu resistance was suppressed in sod1 D cells. Sod1p activity oscillated ~ fivefold during the cell cycle, with peak activity coinciding with peak Cu-resistance. Thus, phenotypic heterogeneity in copper resistance is not stochastic but is driven by the progression of individual cells through the cell cycle and ageing, and is primarily dependent on only Sod1p, out of several gene products that can influence the averaged phenotype. We propose that such heterogeneity provides an important insurance mechanism for organisms; creating subpopulations that are preequipped for varied activities as needs may arise (e.g. when faced with stress), but without the permanent metabolic costs of constitutive expression.