Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.

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Oxygen stress: a regulator of apoptosis in yeast.

Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.

All aerobically growing organisms suffer exposure to oxidative stress, caused by partially reduced forms of molecular oxygen, known as reactive oxygen species (ROS). These are highly reactive and capable of damaging cellular constituents such as DNA, lipids and proteins. Consequently, cells from many different organisms have evolved mechanisms to protect their components against ROS. This review concentrates on the oxidant defence systems of the budding yeast Saccharomyces cerevisiae, which appears to have a number of inducible adaptive stress responses to oxidants, such as H2O2, superoxide anion and lipid peroxidation products. The oxidative stress responses appear to be regulated, at least in part, at the level of transcription and there is considerable overlap between them and many diverse stress responses, allowing the yeast cell to integrate its response towards environmental stress.

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Oxidative stress responses of the yeast Saccharomyces cerevisiae.

In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase Special attention is devoted to physiological studies on S cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption

/pdf/pyruvate-metabolism-in-saccharomyces-cerevisiae-3p6vin11c5.pdf

Pyruvate Metabolism in Saccharomyces cerevisiae

The glutathione- and thioredoxin-dependent reduction systems are responsible for maintaining the reduced environment of the Escherichia coli and Saccharomyces cerevisiae cytosol. Here we examine the roles of these two cellular reduction systems in the bacterial and yeast defenses against oxidative stress. The transcription of a subset of the genes encoding glutathione biosynthetic enzymes, glutathione reductases, glutaredoxins, thioredoxins, and thioredoxin reductases, as well as glutathione- and thioredoxin-dependent peroxidases is clearly induced by oxidative stress in both organisms. However, only some strains carrying mutations in single genes are hypersensitive to oxidants. This is due, in part, to the redundant effects of the gene products and the overlap between the two reduction systems. The construction of strains carrying mutations in multiple genes is helping to elucidate the different roles of glutathione and thioredoxin, and studies with such strains have recently revealed that these two reduction systems modulate the activities of the E. coli OxyR and SoxR and the S. cerevisiae Yap1p transcriptional regulators of the adaptive responses to oxidative stress.

Roles of the Glutathione- and Thioredoxin-Dependent Reduction Systems in the Escherichia Coli and Saccharomyces Cerevisiae Responses to Oxidative Stress

Glutathione (GSH) is an abundant cellular thiol which has been implicated in many cellular processes including protection against xenobiotics, carcinogens and free radicals. Utilization of GSH in both enzymic and non-enzymic defence mechanisms results in its conversion to the oxidized form (GSSG), and it must be recycled to GSH to maintain the high intracellular ratio of GSH to GSSG. Glutathione reductase (GLR) is a flavoenzyme, which catalyses reduction of GSSG to GSH using the reducing power of NADPH. We show that yeast mutants deleted for GLR1, encoding glutathione reductase, lack GLR activity and accumulate increased levels of GSSG. In addition, the glr1 mutant strain was unaffected in the inducible adaptive response to hydrogen peroxide, but showed increased sensitivity to oxidants including both peroxides and superoxide, indicating a requirement for GLR in protection against oxidative stress. Furthermore, GLR1 expression was elevated two to threefold in the presence of oxidants, and regulation was dependent upon the yAP-1 transcriptional activator protein. Thus, GLR1 is one of a growing number of genes involved in the protection of yeast cells against oxidative stress and regulated by yAP-1.

Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation

SUMMARY: Exponential phase cells of the yeast Saccharomyces cerevisiae when treated with a non-lethal concentration of hydrogen peroxide (H2O2; 0·2 mM) for 60 min adapted to become resistant to the lethal effects of a higher dose of H2O2 (2 mM). From studies using cycloheximide to inhibit protein synthesis it appears that protein synthesis is required for maximal induction of resistance but that some degree of protection from the lethal effects of peroxide can be acquired in the absence of protein synthesis. Treatment of cells with 50 μg cycloheximide ml-1 alone led to them acquirng some protection from peroxide. Cells subjected to heat shock became more resistant to 2 mM-H2O2; however, peroxide pretreatment did not confer thermotolerance. L-[35S]Methionine labelling of cells subjected to 0·2 mM-H2O2 stress showed that synthesis of at least ten polypeptides was induced by peroxide treatment. Some of these were also induced in cells subjected to heat shock (23 to 37 °C shift) but the synthesis of at least four polypeptides (45,39·5,38 and 24 kDa) was unique to peroxide-stressed cells. Resistance to peroxide was also inducible in an isogenic petite and an isogenic strain with a mutation in the HAP1 gene, indicating that the adaptive response does not require functional mitochondria.

/pdf/inducibility-of-the-response-of-yeast-cells-to-peroxide-3061u9085h.pdf

Inducibility of the response of yeast cells to peroxide stress.

Exponential phase cells of Saccharomyces cerevisiae treated with the superoxide free-radical generating agent menadione (MD; 0.2 mM) for 60 min adapted to become resistant to the lethal effects of a higher concentration of MD (4 mM). Inhibition of protein synthesis by treatment with cycloheximide totally prevented the adaptation to MD, indicating that this is an inducible response completely dependent on protein synthesis; this differs from the situation with peroxide in which only some of the adaptive response is cycloheximide-sensitive. Cells subjected to heat shock (23 to 37 degrees C) or treatment with hydrogen peroxide (H2O2; 0.2 mM, 60 min) became more resistant to 4 mM-MD; however, MD pretreatment did not induce any thermotolerance or resistance to peroxide. These differences between the response to MD and H2O2 were reflected in the results of L-[35S]methionine labelling studies. Using one-dimensional electrophoresis, only one polypeptide (60 kDa) was seen to be induced by 0.2 mM-MD and this was also induced by heat shock but not peroxide stress. With heat shock or peroxide treatment the induction of at least 10 polypeptides was detected using this approach. Using an isogenic petite strain, it was found that functional mitochondria were needed for conferring full resistance to MD, but that induction of the adaptive response was not dependent on mitochondrial function.

/pdf/saccharomyces-cerevisiae-has-an-inducible-response-to-2v7ymq2kpf.pdf

Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide.

Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase.

The LPD1 gene of Saccharomyces cerevisiae, encoding lipoamide dehydrogenase (LPDH), is subject to catabolite repression The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif Here we report that transcription of LPD1 requires HAP2, HAP3 and HAP4 for release from glucose repression In the wild-type strain, specific activity of LPDH was increased 12-fold by growth on lactate, 10-fold on glycerol and four- to five-fold on galactose or raffinose, compared to growth on glucose In hap2, hap3 and hap4 null mutants, the specific activities of LPDH in cultures grown on galactose and raffinose showed only slight induction above the basal level on glucose medium Similar results were obtained upon assaying for β-galactosidase production in wild-type, or hap2, hap3 or hap4 mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ gene of Escherichia coli and integrated at the URA3 locus Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as it does for the CYC1 gene Site-directed mutagenesis of the putative HAP2/HAP3/HAP4 binding site at −204 relative to the ATG start codon showed that this element was required for full derepression of the LPD1 gene on non-fermetable substrates

Lindsay P. Collinson

Papers

Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation

Inducibility of the response of yeast cells to peroxide stress.

Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide.

Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase.

Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system.