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.


All aerobic organisms are exposed to reactive oxygen species (ROS), such as H2O2, the superoxide anion and the hydroxyl radical, during the course of normal aerobic metabolism or after exposure to radical-generating compounds. Such ROS can cause wide-ranging damage to macromolecules and have been recognized for some time as being associated with various disease processes including cancer, ageing and neurodegenerative disorders (Halliwell and Gutteridge, 1989; Gutteridge, 1993). To protect against oxidant damage, cells contain effective defence mechanisms including enzymes, such as catalase, superoxide dismutase and glutathione peroxidase, and antioxidants, such as glutathione (GSH) and vitamins C and E (reviewed by Yu, 1994). These ROS and the corresponding cellular defence systems activated to protect against them have been studied extensively in the yeast Saccharomyces cerevisiae (Jamieson, 1998).

A number of recent reports have highlighted the key role played by sulphydryl groups (-SH) in the response to oxidative stress and, in particular, the role of the glutathione/glutaredoxin and thioredoxin systems in maintaining the redox homeostasis of the cell (for example, see Demple, 1998; Rietsch and Beckwith, 1998; Stewart et al., 1998). Glutaredoxins and thioredoxins are small heat-stable oxidoreductases that contain two conserved cysteine residues in their active sites (Holmgren, 1989). They were originally identified as hydrogen donors for ribonucleotide reductase, but are also required for a number of metabolic enzymes that form a disulphide as part of their catalytic cycle. Proposed roles include many cellular processes, such as protein folding and regulation, reduction of dehydroascorbate, repair of oxidatively damaged proteins and sulphur metabolism. Glutaredoxins and thioredoxins are structurally similar and have been conserved throughout evolution, particularly in the region of their active site. However, despite considerable functional overlap, they are differentially regulated. The oxidized disulphide form of thioredoxin is reduced directly by NADPH and thioredoxin reductase, whereas glutaredoxin is reduced by glutathione (GSH) using electrons donated by NADPH. Yeast provides an ideal model eukaryote for studying these systems because of the genetic and biochemical tractability of the organism and the availability of null mutants lacking components of both systems. This review describes recent progress made in yeast and how these findings are leading to a better understanding of the functional roles of the GSH/glutaredoxin and thioredoxin systems in eukaryotic growth and metabolism.

Biosynthesis and regulation of GSH

The levels of reduced glutathione (γ-glutamylcysteinylglycine, GSH) are maintained by a complex series of reactions that balance its rate of synthesis and reduction (Fig. 1). The enzymes involved in the biosynthesis (Gsh1, γ-glutamylcysteine synthetase; and Gsh2, glutathione synthetase) and redox regulation (Glr1, glutathione reductase) of GSH have been well characterized (Grant and Dawes, 1996). A homologue of the mammalian enzyme that initiates the breakdown of GSH (γ-glutamyltranspeptidase) has been identified by the yeast genome sequencing project (YLR301w) and experimentally as a dosage-dependent suppresser of a temperature-sensitive cik1 mutant that is involved in microtubule function (Manning et al., 1997). γ-Glutamyltranspeptidase activity has also been detected in yeast and appears be required for supplying growth requirements such as sulphur and nitrogen from intracellular GSH pools (Penninckx and Elskens, 1993). Although the biochemical activities of the γ-glutamyl cycle enzymes (γ-glutamyl-cyclotransferase, cysteinylglycine dipeptidase and 5-oxoprolinase) as described for higher eukaryotes have been detected in yeast, their corresponding genes have not been identified, and little is known regarding GSH recycling via degradation in this organism.

Figure 1.

The yeast glutathione cycle. GSH is synthesized from its constituent amino acids via two ATP-dependent steps. In the first step, γ-glutamylcysteine synthetase, encoded by GSH1, catalyses the formation of the dipeptide γ-glutamylcysteine (γ-GluCys) from glutamic acid and cysteine and, in the second step, glutathione synthetase (GSH2) catalyses the ligation of the dipeptide with glycine. The first step in GSH synthesis is the rate-limiting step and is feedback inhibited by the end-product GSH. GSH is oxidized to GSSG by reaction with free radicals or in reactions catalysed by glutathione peroxidase (encoded by GPX1-3) or glutathione S-transferase (encoded by GTT1 and GTT2). GSH is regenerated in an NADPH-dependent reaction catalysed by glutathione reductase (encoded by GLR1). The factors that control the degradation of GSH/GSSG are unknown in yeast.

The glutaredoxin system

Yeast contains two glutaredoxin genes, designated GRX1 and GRX2 (Fig. 2), which share 40–52% identity and 61–76% similarity with those from bacterial and mammalian species (Luikenhuis et al., 1997). Strains deleted for both GRX1 and GRX2 are viable but lack heat-stable oxidoreductase activity, using β-hydroxyethyl disulphide as a model disulphide substrate. Surprisingly, given the high degree of homology between Grx1 and Grx2 (64% identity), Grx2 accounts for the majority of this activity in vivo. In addition, the grx1 mutant is sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacks GRX2 is sensitive to hydrogen peroxide. This difference in oxidant sensitivity may reflect differences in the substrate proteins regulated by Grx1 and Grx2 or in their ability to detoxify ROS-mediated damage and is consistent with the idea that the two glutaredoxin isoforms play distinct roles during normal growth and stress conditions.

Figure 2.

The GSH/glutaredoxin and thioredoxin systems. Yeast contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2). The oxidized disulphide form of thioredoxin is reduced directly by NADPH and thioredoxin reductase. In contrast, oxidized glutaredoxin is reduced by glutathione (GSH), and oxidized GSSG is in turn reduced in an NADPH-dependent reaction catalysed by glutathione reductase. The redox status of GSH may provide a functional link between the GSH/glutaredoxin and thioredoxin systems, as thioredoxins function along with Glr1 to maintain the high intracellular GSH–GSSG ratio. It is not known whether the redox status of the GSH/glutaredoxin system affects the thioredoxin system. Thioredoxins act as hydrogen donors for 3′-phosphoadenosine 5′-phosphosulphate reductase (PAPS reductase) and thioredoxin peroxidases (Trx peroxidase). Both glutaredoxins and thioredoxins are believed to act as hydrogen donors for ribonucleotide reductase. No glutaredoxin-specific targets have yet been identified.

A new family of glutaredoxin-related proteins has been identified in yeast (GRX3–5) that is conserved throughout evolution from bacterial to mammalian species (Rodriguez-Manzaneque et al., 1999). These glutaredoxin-like proteins differ from classical glutaredoxins in that they contain a single cysteine residue at their putative active sites. Thus, they would be unlikely to substitute for glutaredoxins or thioredoxins as disulphide reductases with substrates such as ribonucleotide reductase, as this requires a dithiol mechanism (Bushweller et al., 1992). Grx3, 4 and 5 may therefore be required to reduce protein-mixed disulphides, which are formed during exposure to ROS, a reaction that proceeds via a monothiol mechanism (Bushweller et al., 1992; Rodriguez-Manzaneque et al., 1999). Loss of GRX3, 4 or 5 results in decreased oxidoreductase activity, and the triple mutant lacking all three isoforms is inviable. In addition, the grx5 mutant is particularly sensitive to oxidative stress induced by H2O2 and the superoxide anion, shows elevated levels of oxidative protein damage and is slow growing. Interestingly, a grx2 grx5 mutant is inviable, indicating that there may be some overlapping function between the two classes of glutaredoxins that may be related to their common activity as GSH-dependent oxidoreductases in protection against protein oxidative damage. The available genetic and biochemical data indicate that the five glutaredoxin genes in yeast have complicated and overlapping functions.

The thioredoxin system

Yeast contains two genes encoding cytoplasmic thioredoxins (TRX1 and TRX2;Fig. 2) that are dispensable under normal growth conditions (Gan, 1991; Muller, 1991). Deletion of both TRX1 and TRX2 affects the cell cycle, resulting in a prolonged S phase and a shortened G1 interval, which do not result from alterations in the levels of deoxyribonucleotides (Muller, 1991). Thioredoxin mutants (trx1 trx2) are defective in sulphate assimilation, consistent with a role as hydrogen donors for 3′-phosphoadenosine 5′-phosphosulphate reductase, the enzyme that converts 3′-phosphoadenosine 5′-phosphosulphate (PAPS) to sulphite (Muller, 1991). Like glutaredoxins, Trx2 functions in protection against ROS, as trx2 mutants are sensitive to H2O2 (Kuge and Jones, 1994) and resistant to the thiol oxidant diamide (Muller, 1996). Additionally, the yAP-1 transcriptional activator protein, which regulates a number of oxidative stress-responsive genes, is constitutively activated in a trx1 trx2 mutant (Izawa et al., 1999).

Thioredoxin reductase was originally purified based on its ability to reduce thioredoxin peroxidase (TSA1) in the presence of thioredoxin (Chae et al., 1994). Antibodies raised against the purified protein were used to clone the corresponding gene, which shares 51% identity and 69% similarity with the Escherichia coli enzyme. Subsequently, TRR1 has been identified as a gene that affects the transcription of cell cycle-regulated genes at the G1/S boundary (Machado et al., 1997) and as a gene that affects the activity of the p53 tumour suppresser gene in yeast (Pearson and Merril, 1998). Strains deleted for TRR1 are hypersensitive to hydrogen peroxide, temperature sensitive for growth and have an auxotrophic requirement for methionine (Machado et al., 1997; Pearson and Merril, 1998).

A mitochondrial thioredoxin system has also been identified in yeast, including a thioredoxin (TRX3) and thioredoxin reductase (TRR2), which function in protection against oxidative stress generated during respiratory metabolism (Pedrajas et al., 1999). However, given the lethality of strains deleted for GRX1, GRX2, TRX1 and TRX2, it does not appear that this mitochondrial system can substitute for the cytoplasmic thioredoxin or glutaredoxin systems (Draculic et al., 2000).

A single glutaredoxin or thioredoxin is essential for viability in yeast

The first indication of a functional link between the thioredoxin and GSH/glutaredoxin systems in yeast came from the identification of a glutathione reductase (GLR1) null mutant in a genetic screen for mutations that confer a requirement for thioredoxins (Muller, 1996). Mutants lacking both TRX1 and TRX2 are inviable in the absence of GLR1, indicating that thioredoxin mutants require the ability to reduce GSH for survival. Limited growth of the triple trx1 trx2 glr1 mutant could be restored using anaerobic conditions, indicating that oxidative stress may play a role in the lethality of this strain. This synthetic lethality does not appear to arise from a defect in deoxyribonucleotide synthesis, as the addition of reduced GSH does not rescue the trx1 trx2 glr1 mutant. A further overlap between the two systems is evident from the finding that loss of both TRX1 and TRX2 results in elevated levels of GSSG, indicating a link between the thioredoxin system and the redox status of GSH in the cell (Muller, 1996). Deletion of GLR1 in the trx1 or trx2 mutant further exacerbates this elevation in GSSG levels, indicating that thioredoxins may normally function along with Glr1 to maintain the high intracellular GSH–GSSG ratio. Thus, the redox status of GSH may provide a functional link between the GSH/glutaredoxin and thioredoxin systems (Fig. 2). In this view, any condition that affects the thioredoxin redox balance shifting it towards a more oxidized form may also affect the redox state of GSH and, hence, the activity of the glutaredoxin system. It is not known whether the redox state of glutaredoxins can affect the thioredoxin system, but deletion of GRX1 or GRX2 does not affect the redox state or levels of GSH (Luikenhuis et al., 1997).

Thioredoxins and glutaredoxins have been conserved throughout evolution, particularly in the region of their active sites. Not surprisingly, therefore, considerable functional redundancy has been identified between the two systems, including activity towards ribonucleotide reductase, PAPS reductase and plasma glutathione peroxidase (Holmgren, 1989; Bjornstedt et al., 1994; Lillig et al., 1999). A single functional disulphide reductase system is essential for viability in bacterial species, as mutants lacking thioredoxin reductase (trxB) and either glutathione reductase (gor) or glutathione synthetase (gshA) are inviable (Prinz et al., 1997). This may indicate an essential function in the reduction of enzymes such as ribonucleotide reductase or, alternatively, in reducing non-native disulphide bonds that may be formed in the cytoplasm during aerobic growth (Prinz et al., 1997). Similarly, the quadruple trx1 trx2 grx1 grx2 yeast mutant is inviable, and a single glutaredoxin or thioredoxin is necessary and sufficient for growth (Draculic et al., 2000). The reason for this essential requirement for a glutaredoxin or thioredoxin is unknown at present. It remains to be established which of the yeast glutaredoxin or thioredoxin isoenzymes can function in the reduction of ribonucleotides, but it seems likely that both systems are active, as deoxynucleotide triphosphate levels are unaltered in a trx1 trx2 mutant (Muller, 1994), and stains lacking thioredoxins, but containing a single glutaredoxin, are viable (Draculic et al., 2000). Similarly, any role for the yeast glutaredoxins or thioredoxins in disulphide bond formation and isomerization remains to be established, but seems likely given the bacterial requirement for thioredoxin in these processes (reviewed by Debarbieux and Beckwith, 1999; Frand et al., 2000).

Role of the glutathione/glutaredoxin system in defence against oxidative stress

GSH is an important antioxidant, as yeast strains lacking GSH or altered in their GSH redox state are sensitive to oxidative stress induced by peroxides and the superoxide anion, as well as the toxic products of lipid peroxidation (Izawa et al., 1995; Grant et al., 1996a, b; Stephan and Jamieson, 1996; Turton et al., 1997). Exponential phase cells grown on rich YEPD media under normal aerobic conditions have a high redox ratio (GSH–GSSG) of ≈ 11–16:1 (Grant et al., 1996b; Muller, 1996), indicating that most of the intracellular glutathione is maintained in a reduced (GSH) form. Exposure of yeast cells to H2O2 causes a reduction in GSH levels, as well as a shift in the redox balance towards the more oxidized form (Grant et al., 1998). Of the total glutathione detected, most is present in a free (unbound) form either intracellularly (58%) or extracellularly (39%). In response to a challenge with H2O2, oxidized GSSG, protein-bound (GSSP) and extracellular glutathione are all elevated in parallel with the reduction in intracellular GSH levels (Grant et al., 1998). This increase in oxidized GSSG is consistent with the role of GSH as both a free radical scavenger and a cofactor for various antioxidant enzymes, including glutathione peroxidases, glutathione S-transferases and glutaredoxins.

Glutathione peroxidase

Both selenium-dependent and -independent glutathione peroxidases have been described that catalyse the breakdown of hydroperoxides using GSH as a reductant. Published data concerning their presence in microorganisms have been somewhat conflicting (reviewed by Penninckx and Elskens, 1993). However, recent studies have confirmed the presence of glutathione peroxidases in yeast. Glutathione peroxidase activity towards both H2O2 and organic peroxides has been detected, and three gene products (GPX1, GPX2 and GPX3) with significant homology (36% identity) to mammalian glutathione peroxidases have been identified from the yeast genome (Inoue et al., 1999). The greatest sequence homology with mammalian glutathione peroxidases is found in the region around their active sites, but selenocysteine (encoded by TGA) appears to be replaced by a cysteine residue in the yeast proteins. Mutants deleted for GPX1-3 (both singly and in combination) are viable and grow at the same rate as the wild-type strain under non-stress conditions (Inoue et al., 1999). Disruption of GPX3 alone results in sensitivity to hydrogen peroxide and tert-butyl hydroperoxide, and Gpx3 appears to account for the majority of glutathione peroxidase activity in the cell (using tert-butyl hydroperoxide as a substrate). In addition, the basal levels of GPX3 are constitutively high, whereas the expression of GPX1 and GPX2 is regulated by different stress conditions. Thus, the different glutathione peroxidase isoforms appear to play distinct roles in the cell, but the identities of their intracellular targets remain unknown.

Glutathione conjugation and transport

Many lipophilic compounds are conjugated to GSH by the action of glutathione S-transferases before their removal from the cytosol by ATP-dependent GS-X pumps. The Ycf1 GS-X pump was first identified based on its ability to confer resistance to the heavy metal cadmium when present at an elevated gene dosage (Szczypka et al., 1994). Ycf1 is a member of the ATP-binding cassette (ABC) protein superfamily and shares extensive homology with the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). It is a functional homologue of MRP1, active in the uptake of glutathione S-conjugates into the vacuole (Ze-Sheng et al., 1996). Furthermore, strains lacking YCF1 are sensitive to 1-chloro-2,4-dinitrobenzene (CDNB) and cadmium, indicating a function in the detoxification of S-conjugated xenobiotics. Two genes encoding functional glutathione S-transferases, designated GTT1 and GTT2, have been identified in yeast (Choi et al., 1998). Purified recombinant Gtt1 and Gtt2 are active in a glutathione S-transferase assay using CDNB as a model substrate. However, Gtt1 and Gtt2 share limited sequence homology with each other (11% identity, 32% similarity) as well as with glutathione S-transferases from other species, with the most conserved residues corresponding to the GSH binding site. Subcellular fractionation experiments indicate that Gtt1 is associated with the endoplasmic reticulum and, in addition, its expression is increased in response to stress conditions. Strains deleted for GTT1 and GTT2 are viable, but show no increased sensitivity to ROS, indicating that they are not required for protection against oxidative stress. The gtt1 gtt2 mutant is sensitive to heat stress during stationary phase growth but, surprisingly, given the sensitivity of ycf1 mutants to the GST substrate CDNB, is unaffected in resistance to CDNB (Choi et al., 1998).

Protein–glutathione mixed disulphides

Cysteine residues are among the most easily oxidized residues in proteins, resulting in intermolecular protein cross-linking and enzyme inactivation (Coan et al., 1992). Such irreversible oxidation events can be prevented by protein S-thiolation, in which protein -SH groups form mixed disulphides with low-molecular-weight thiols such as GSH (Thomas et al., 1995; Cotgreave and Gerdes, 1998). Protein S-thiolation is reversible, and dethiolation can occur via direct reduction by GSH. In addition, protein dethiolation can be catalysed in vitro by glutaredoxins and thioredoxins (Thomas et al., 1995), and a recent study has indicated a correlation between protein–SSG reduction and glutaredoxin activity in mammalian cells (Chrestensen et al., 2000). Protein S-thiolation may, therefore, represent a novel form of post-translational modification that can regulate protein activity in response to growth or other cellular signals and, for example, has been implicated in the regulation of the HIV-1 protease under the control of glutaredoxin (Davis et al., 1997) and in regulating ubiquitin-conjugating enzymes in bovine retina cells (Obin et al., 1998).

The exact role of thiolation in protection against ROS-mediated damage is unclear at present, as are the consequences of this process for cell growth and activity. A number of S-thiolated proteins have been detected in studies using human endothelial cells and murine macrophages, including carbonic anhydrase, actin, creatine kinase and glyceraldehyde 3-P dehydrogenase (GAPDH). There does not appear to be any unifying feature to these proteins apart from the fact that they are relatively abundant in mammalian cells (reviewed by Thomas et al., 1995; Cotgreave and Gerdes, 1998). GAPDH has also been identified as the major target of protein S-thiolation in yeast (Grant et al., 1999), in which the process is tightly regulated as, despite a high degree of sequence homology (98% similarity, 96% identity), the Tdh3, but not the Tdh2, GAPDH isoenzyme is S-thiolated. Protein S-thiolation results in a reversible inhibition of Tdh3 enzyme activity, which appears to be physiologically important, as yeast cells lacking TDH3 are sensitive to a challenge with a lethal dose of H2O2. Any role for the yeast glutaredoxins in protein S-thiolation or dethiolation remains to be established, but seems likely given their activity as GSH-dependent oxidoreductases.

Role of the thioredoxin system in defence against oxidative stress

The first yeast thioredoxin peroxidase was characterized as a thiol-specific antioxidant (TSA) that could provide protection against a thiol-containing oxidation system. Tsa1 was subsequently shown to possess peroxidase activity towards hydrogen peroxide and alkyl hydroperoxides in the presence of the thioredoxin and thioredoxin reductase reducing system. Yeast strains lacking TSA1 are viable and are sensitive to oxidative stress induced by the superoxide anion and peroxides including hydrogen peroxide and tert-butyl hydroperoxide (Chae et al., 1993). A second type of thioredoxin peroxidase (type II, Tsa2) has been purified from yeast, which shows limited sequence homology to TSA1 (Jeong et al., 1999). Kinetic analysis indicates that it preferentially reduces alkyl hydroperoxides compared with hydrogen peroxide, the reverse of the preference shown by Tsa1. The reaction mechanism appears to involve the formation of an intermolecular disulphide bond between two protein molecules that can be reduced by thioredoxin but not by GSH. This type II thioredoxin peroxidase has also been isolated in two independent genetic screens designed to isolate novel antioxidants. First, AHP1 (YLR109w) was isolated in a screen for genes that restore tert-butyl hydroperoxide resistance to a strain deleted for SKN7 (Lee et al., 1999a) and, secondly, YLR109 was isolated using a C35S thioredoxin mutant designed to trap proteins that form a disulphide linkage with thioredoxin (Verdoucq et al., 1999). Analysis of mutants lacking TSA1 or AHP1 indicates that TSA1 is more involved in H2O2 resistance compared with AHP1, which appears to be specific for organic peroxides (Lee et al., 1999a).

A large number of thioredoxin peroxidase isoforms have been detected in mammalian cells and, similarly, three additional enzymes have been identified from the yeast genome sequencing project (Park et al., 2000). Enzyme analysis has confirmed that all the gene products display thioredoxin peroxidase activity, and mutating their putative functional cysteine residues completely inactivates antioxidant activity. Various intracellular localizations have been determined including cytoplasmic (Tsa1, cTpx2/YDR453c, Tsa2/Ahp1/YLR109), mitochondrial (mTpx/YBL064c) and nuclear (nTpx/YIL010w) (Park et al., 2000). Transcript analysis and lacZ transcriptional fusion assays indicate that Tsa1 and Tsa2 (Ahp1/YLR109) are the most abundant isoforms present during normal aerobic growth. In addition, the levels of cytoplasmic and mitochondrial Tpxs are all induced by oxidative stress, including hydrogen peroxide, diamide and transfer from anaerobic to aerobic growth conditions. Interestingly, mutants lacking YDR453c display a severe slow growth phenotype resulting from an accumulation of cells in the G1 phase of the cell cycle. This is in contrast to strains deleted for both TRX1 and TRX2, which results in a prolonged S phase and a shortened G1 interval in the cell cycle (Muller, 1991). The mitochondrial protein encoded by YBL064c has recently been identified as the first 1-cys peroxiredoxin from yeast and named Prx1 (Pedrajas et al., 2000). It is active as a thioredoxin peroxidase, but appears to use the mitochondrial thioredoxin system as an electron donor to protect against oxidative stress generated by mitochondrial metabolism.

Regulation in response to stress conditions

In response to stress conditions, yeast cells upregulate the synthesis of a number of protective molecules including components of both the GSH/glutaredoxin and the thioredoxin systems. This forms the basis of an inducible adaptive response, in which, for example, cells treated with a low dose of oxidant can adapt to become resistant to a subsequent and otherwise lethal treatment. A number of stress-responsive control mechanisms have been implicated in these systems, including the yAP-1 and Skn7 transcriptional regulators and the general stress response/STRE pathway (Table 1).

Table 1. Regulation of the GSH/glutaredoxin and thioredoxin systems in response to stress conditions.
GeneStress conditionaControl sitebReference
  • a . Denotes where expression is known to be induced by a particular stress condition. P, peroxides; Su, O 2 (menadione or paraquat); D, diamide; Os, osmotic stress; Hs, heat shock; Nf, non-fermentable carbon source; St, stationary phase growth.

  • b

    . ARE, yAP-1-response element; STRE, stress-response element.

GSH/GRX system
 GSH1P, SuARE Stephen et al. (1995); Wu and Moye-Rowley (1994)
 GSH2P, HsARE Sugiyama et al. (2000)
 GLR1P, D, StARE Grant et al. (1996a, b)
 GRX1P, Su, D, Os, Hs, Nf, StSTRE Grant et al. (2000)
 GRX2P, Su, D, Os, Hs, Nf, StSTRE Grant et al. (2000)
 GPX1Su, Hs  Inoue et al. (1999)
 GPX2P, Su,ARE Inoue et al. (1999)
 GTT1St, OsARE, STRE Choi et al. (1998)
 YCF1Not knownARE Wemmie et al. (1994)
TRX system
 TRX2P, Su, DARE, SKN7 Kuge and Jones (1994); Lee et al. (1999b); Morgan et al. (1997)
 TRR1P, DARE, SKN7 Lee et al. (1999b) ; Morgan et al. (1997)
 TSA1P, D, StARE, SKN7 Charizanis et al. (1999); Lee et al. (1999b); Park et al. (2000)
 AHP1/YLR109P, D, StARE, SKN7 Lee et al. (1999b); Park et al. (2000)
 YDR453cP, D, St, OsARE, SKN7 Lee et al. (1999b); Park et al. (2000)
 YBL064cP, D, St, Os  Park et al. (2000)

The synthesis and reduction of GSH appears to be primarily regulated by the yAP-1 transcription factor, as GSH1, GSH2 and GLR1 contain yAP-1 binding sites (ARE), and their expression is elevated in response to ROS (Wu and Moye-Rowley, 1994; Stephen et al., 1995; Grant et al., 1996b; Sugiyama et al., 2000). Thus, in response to oxidative stress, cells can elevate GSH levels by means of both increased synthesis and recycling oxidized GSSG to its reduced form. Similarly, ARE elements have been identified in GSH-dependent antioxidants including GPX2, GTT1 and YCF1, all of which are upregulated in response to stress conditions (Wemmie et al., 1994; Choi et al., 1998; Inoue et al., 1999).

In contrast to the GSH pathway, the expression of thioredoxin-related genes is subject to regulation by both the yAP-1 and Skn7 (Pos9) transcriptional activator proteins. The synthesis and reduction of thioredoxin through TRX2 and TRR1 is induced in response to oxidative stress conditions via the co-operative action of the yAP-1 and Skn7 transcription factors (Kuge and Jones, 1994; Morgan et al., 1997). Similarly, Skn7-dependent, yAP-1-regulated genes include the thioredoxin peroxidase isoenzymes TSA1, TSA2, YDR453c and YBL064c (Charizanis et al., 1999; Lee et al., 1999b; Park et al., 2000).

Thus, the regulation of both the thioredoxin system and the GSH pathway requires the yAP-1 transcriptional activator protein, but can be separated based on the requirement for Skn7. In contrast, the expression of both GRX1 and GRX2 is activated by the high-osmolarity glycerol pathway (HOG) and negatively regulated by the Ras–protein kinase A pathway (PKA) via stress-responsive STRE elements (Grant et al., 2000). Expression of both genes is induced in response to various stress conditions, including oxidative, osmotic, heat, stationary phase growth and growth on non-fermentable carbon sources. GRX1 contains a single STRE element and is induced to significantly higher levels compared with GRX2 after heat and osmotic shock. GRX2 contains two STRE elements and is rapidly induced in response to ROS and upon entry into stationary phase growth. These data support the idea that the two yeast glutaredoxin isoforms play distinct roles during normal cellular growth and in response to stress conditions. Similarly, a putative STRE element has been identified in GTT1, which may account for the increase in expression seen in response to stationary phase growth and osmotic shock (Choi et al., 1998).

Future directions

Although the yeast system has enabled a clearer understanding of the overlapping functions of the GSH/glutaredoxin and thioredoxin systems, important questions remain to be addressed. It is clear that both systems have common functions and regulatory mechanisms, but their exact role(s) in cellular metabolism and redox homeostasis remains to be established. In particular, the essential requirement for a single glutaredoxin or thioredoxin is unknown, and no targets for the yeast glutaredoxins have yet been identified. The identification and isolation of the components comprising both systems should facilitate studies that are aimed at answering these important questions. Establishing how these systems can regulate such a diverse range of functions including cell growth, nutrient assimilation and stress responses and, in turn, how the regulation of these various functions is achieved will be a major challenge.


The author would like to thank Ian Dawes (UNSW) and the members of his laboratory for many helpful discussions. Work in the author's laboratory is supported by the BBSRC and the Wellcome Trust.