Chris M. Grant

Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions.

Sulphydryl groups (‐SH) play a remarkably broad range of roles in the cell, and the redox status of cysteine residues can affect both the structure and the function of numerous enzymes, receptors and transcription factors. The intracellular milieu is usually a reducing environment as a result of high concentrations of the low‐molecular‐weight thiol glutathione (GSH). However, reactive oxygen species (ROS), which are the products of normal aerobic metabolism, as well as naturally occurring free radical‐generating compounds, can alter this redox balance. A number of cellular factors have been implicated in the regulation of redox homeostasis, including the glutathione/glutaredoxin and thioredoxin systems. Glutaredoxins and thioredoxins are ubiquitous small heat‐stable oxidoreductases that have proposed functions in many cellular processes, including deoxyribonucleotide synthesis, repair of oxidatively damaged proteins, protein folding and sulphur metabolism. This review describes recent findings in the lower eukaryote Saccharomyces cerevisiae that are leading to a better understanding of their role in redox homeostasis in eukaryotic cell metabolism.

GLUTATHIONE IS AN ESSENTIAL METABOLITE REQUIRED FOR RESISTANCE TO OXIDATIVE STRESS IN THE YEAST SACCHAROMYCES CEREVISIAE

Glutathione (GSH) is an abundant cellular thiol which has been implicated in numerous cellular processes and in protection against stress caused by xenobiotics, carcinogens and radiation. Our experiments address the requirement for GSH in yeast, and its role in protection against oxidative stress. Mutants which are unable to synthesis GSH due to a gene disruption inGSH 1, encoding the enzyme for the first step in the biosynthesis of GSH, require exogenous GSH for growth under non-stress conditions. Growth can also be restored with reducing agents containing a sulphydryl group, including dithiothreitol, β-mercaptoethanol and cysteine, indicating that GSH is essential only as a reductant during normal cellular processes. In addition, theGSH 1-disruption strain is sensitive to oxidative stress caused by H2O2 and tert-butyl hydroperoxide. The requirement for GSH in protection against oxidative stress is analogous to that in higher eukaryotes, but unlike the situation in bacteria where it is dispensable for growth during both normal and oxidative stress conditions.

The Response to Heat Shock and Oxidative Stress in Saccharomyces cerevisiae

A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.

Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H2O2 in the yeast Saccharomyces cerevisiae. Our data show that H2O2 causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the α-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H2O2 in a gcn2 mutant. Polysome analysis indicated that H2O2 causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H2O2 stress. For some candidate mRNAs, we show that a low concentration of H2O2 results in increased protein production. In contrast, a high concentration of H2O2 promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae

The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, a process by which protein SH groups form mixed disulphides with low-molecular-mass thiols such as glutathione. We report here the target proteins which are modified in yeast cells in response to H(2)O(2). In particular, a range of glycolytic and related enzymes (Tdh3, Eno2, Adh1, Tpi1, Ald6 and Fba1), as well as translation factors (Tef2, Tef5, Nip1 and Rps5) are identified. The oxidative stress conditions used to induce S-thiolation are shown to inhibit GAPDH (glyceraldehyde-3-phosphate dehydrogenase), enolase and alcohol dehydrogenase activities, whereas they have no effect on aldolase, triose phosphate isomerase or aldehyde dehydrogenase activities. The inhibition of GAPDH, enolase and alcohol dehydrogenase is readily reversible once the oxidant is removed. In addition, we show that peroxide stress has little or no effect on glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, the enzymes that catalyse NADPH production via the pentose phosphate pathway. Thus the inhibition of glycolytic flux is proposed to result in glucose equivalents entering the pentose phosphate pathway for the generation of NADPH. Radiolabelling is used to confirm that peroxide stress results in a rapid and reversible inhibition of protein synthesis. Furthermore, we show that glycolytic enzyme activities and protein synthesis are irreversibly inhibited in a mutant that lacks glutathione, and hence cannot modify proteins by S-thiolation. In summary, protein S-thiolation appears to serve an adaptive function during exposure to an oxidative stress by reprogramming metabolism and protecting protein synthesis against irreversible oxidation.

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

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.

Metabolic reconfiguration is a regulated response to oxidative stress

A new study reveals that, in response to oxidative stress, organisms can redirect their metabolic flux from glycolysis to the pentose phosphate pathway, the pathway that provides the reducing power for the main cellular redox systems. This ability is conserved between yeast and animals, showing its importance in the adaptation to oxidative stress.

Role of thioredoxins in the response of Saccharomyces cerevisiae to oxidative stress induced by hydroperoxides

Glutaredoxins 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 medi‐ating 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 protein‐bound levels of the low‐molecular‐weight antioxidant glutathione.

Differential Protein S-Thiolation of Glyceraldehyde-3-Phosphate Dehydrogenase Isoenzymes Influences Sensitivity to Oxidative Stress

ABSTRACT The irreversible oxidation of cysteine residues can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulfides with low-molecular-weight thiols such as glutathione. We report here the identification of glyceraldehyde-3-phosphate dehydrogenase as the major target of protein S-thiolation following treatment with hydrogen peroxide in the yeastSaccharomyces cerevisiae. Our studies reveal that this process is tightly regulated, since, surprisingly, despite a high degree of sequence homology (98% similarity and 96% identity), the Tdh3 but not the Tdh2 isoenzyme was S-thiolated. The glyceraldehyde-3-phosphate dehydrogenase enzyme activity of both the Tdh2 and Tdh3 isoenzymes was decreased following exposure to H2O2, but only Tdh3 activity was restored within a 2-h recovery period. This indicates that the inhibition of the S-thiolated Tdh3 polypeptide was readily reversible. Moreover, mutants lacking TDH3 were sensitive to a challenge with a lethal dose of H2O2, indicating that the S-thiolated Tdh3 polypeptide is required for survival during conditions of oxidative stress. In contrast, a requirement for the nonthiolated Tdh2 polypeptide was found during exposure to continuous low levels of oxidants, conditions where the Tdh3 polypeptide would be S-thiolated and hence inactivated. We propose a model in which both enzymes are required during conditions of oxidative stress but play complementary roles depending on their ability to undergo S-thiolation.

Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae

Yeast strains that lack mitochondrial function are sensitive to oxidative stress caused by reactive oxygen species (ROS). Specifically, rho0 mutants that lack mitochondrial DNA, and strains deleted for the nuclear genes COX6 and COQ3 that are required for function of the respiratory electron transport chain, were sensitive to H2O2. In addition, treatment with mitochondrial inhibitors including antimycin A, oligomycin, potassium cyanide and sodium azide increased sensitivity to H2O2. The mechanism does not appear to depend on the antioxidant status of the cell since respiratory‐deficient strains were able to mount an inducible adaptive response to H2O2. We suggest that the oxidant sensitivity is due to a defect in an energy‐requiring process that is needed for detoxification of ROS or for the repair of oxidatively damaged molecules.