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.
Many 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 the maintenance of the redox homeostasis of the cell (Rietsch and Beckwith, 1998; Stewart et al., 1998; Grant, 2001). Glutaredoxins and thioredoxins are small heat-stable oxidoreductases that contain two conserved cysteine residues at their active sites (Holmgren, 1989). They were originally identified as hydrogen donors for ribonucleotide reductase, but are also active towards a number of metabolic enzymes, which form a disulphide as part of their catalytic cycle. Considerable functional redundancy has been identified between the two systems, including activity towards ribonucleotide reductase, 3′-phosphoadenosine 5′-phosphosulphate reductase (PAPS reductase) and plasma glutathione peroxidase (Holmgren, 1989; Bjornstedt et al., 1994; Lillig et al., 1999). Not surprisingly, therefore, a single functional disulphide reductase system is essential for viability. In bacterial species, mutants lacking thioredoxin reductase (trxB) and either glutathione reductase (gor) or glutathione synthetase (gshA) are inviable (Prinz et al., 1997). Similarly, yeast mutants lacking all cytoplasmic thioredoxins or glutaredoxins are inviable, and a single glutaredoxin or thioredoxin is necessary and sufficient for growth (Draculic et al., 2000). Thioredoxins and glutaredoxins are also 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 (Holmgren, 1990).
Yeast contains two genes encoding cytoplasmic thioredoxins (TRX1 and TRX2), which are dispensable during normal growth conditions (Gan, 1991; Muller, 1991). Deletion of both TRX1 and TRX2 affects the cell cycle, resulting in a prolonged S phase and shortened G1 interval (Muller, 1991). This cell cycle defect does not result from alterations in the levels of deoxyribonucleotides, but does require a redox-dependent function (Muller, 1995). Thioredoxins are also active as hydrogen donors for PAPS reductase, the enzyme that converts 3′-phosphoadenosine 5′-phosphosulphate (PAPS) to sulphite and, hence, trx1 trx2 mutants are unable to grow on media containing sulphate as the sole source of metabolic sulphur (Muller, 1991). Yeast also contains two genes encoding cyto-plasmic glutaredoxins (GRX1 and GRX2), which are dispensable during normal growth conditions (Luikenhuis et al., 1997). Unlike the thioredoxins, however, loss of glutaredoxins does not affect the cell cycle, nor does it result in any detectable aberrant growth phenotype.
As in other species, yeast thioredoxins and glutaredoxins are active as antioxidants and play key roles in protection against oxidative stress induced by various reactive oxygen species (ROS). TRX2 was originally identified as a target gene of the yAP-1 transcriptional activator protein, which regulates the expression of a large number of antioxidants in response to oxidative stress (Kuge and Jones, 1994; Toone and Jones, 1999). Furthermore, TRX2 was shown to be essential for yAP-1-mediated resistance to hydroperoxides (Kuge and Jones, 1994). Thioredoxins are also required for the detoxification of ROS through their reactivity with thioredoxin peroxidases. A large number of thioredoxin peroxidase isoforms have been identified in yeast, includ-ing cytoplasmic (Tsa1, cTpx2/YDR453c, Tsa2/Ahp1/YLR109), mitochondrial (mTpx/YBL064c) and nuclear (nTpx/YIL010w) (Chae et al., 1993; Jeong et al., 1999; Verdoucq et al., 1999; Park et al., 2000; Pedrajas et al., 2000). Enzyme analysis has confirmed that all the gene products display thioredoxin peroxidase activity, but their exact intracellular roles and targets remain unclear (Park et al., 2000). Strains deleted for both GRX1 and GRX2 lack heat-stable oxidoreductase activity using β-hydroxyethylene disulphide as a model disulphide substrate (Luikenhuis et al., 1997). Interestingly, a grx1 mutant was found to be sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Thus, Grx1 and Grx2 appear to function differently in yeast, but it is not yet known what their respective roles or substrates may be (Grant et al., 2000).
We have shown previously that the quadruple grx1 grx2 trx1 trx2 mutant is inviable, but strains either containing a single glutaredoxin or thioredoxin gene (i.e. grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) or lacking any pair of genes (trx1 grx1, trx1 grx2, trx2 grx1, trx2 grx2) are viable (Draculic et al., 2000). In the present study, we have used these mutant strains to compare the roles of thioredoxins and glutaredoxins as antioxidants. Thioredoxins were found to be more important than glutaredoxins in mediating resistance to oxidative stress caused by hydroperoxides. Trx1 and Trx2 appear to be functionally redundant as antioxidants, and are particularly required during stationary phase growth. In particular, thioredoxins were required to act as cofactors for the thioredoxin peroxidase Tsa1, as well as to regulate the redox state and levels of protein-bound glutathione in response to oxidative stress.
Comparison of the roles of thioredoxins and glutaredoxins in mediating resistance to oxidative stress
The availability of strains lacking combinations of glutaredoxins and thioredoxins (Draculic et al., 2000) has enabled us to examine the requirement for each glutaredoxin or thioredoxin individually in the cell. In particular, we compared strains deleted for thioredoxins (trx1, trx2, trx1 trx2), glutaredoxins (grx1, grx2, grx1 grx2) and combinations of both (grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) for resistance to oxidative stress. Strains were grown to stationary phase and spotted onto YEPD plates containing various concentrations of oxidants (Fig. 1A). On 4 mM H2O2, strains lacking glutaredoxins, both singly and in combination, as well as strains lacking TRX1 showed wild type-levels of resistance. In contrast, the trx2 and trx1 trx2 mutants were hypersensitive to H2O2 and did not grow. In addition, all triple mutants that were deleted for TRX2 (trx1 trx2 grx1, trx1 trx2 grx2, trx2 grx1 grx2) were sensitive to H2O2. Interestingly, the strain containing TRX2 alone (trx1 grx1 grx2) was resist-ant to H2O2, indicating that Trx2 is the most important thioredoxin or glutaredoxin in determining resistance to H2O2. Similar results were obtained with tert-butyl hydroperoxide. Strains lacking TRX2 were sensitive to this oxidant, whereas strains containing the TRX2 gene displayed wild-type levels of resistance (Fig. 1A). None of the mutants was sensitive to the superoxide anion generated by the redox cycling agent menadione (data not shown). Thus, thioredoxins appear to be more important than glutaredoxins in mediating resistance to hydro-peroxides, with Trx2 playing the predominant role.
To test directly whether both Trx1 and Trx2 could mediate resistance to ROS, we examined whether overexpression of TRX1 and TRX2 affected oxidant sensitivity. Overexpression of TRX1 and TRX2 was found to increase markedly the resistance of a wild-type strain to both H2O2 and tert-butyl hydroperoxide (Fig. 1B). In comparison, overexpression of GRX1 and GRX2 resulted in a modest increase in resistance to H2O2, but did not affect tert-butyl hydroperoxide resistance (Fig. 1B). No effects were seen with thioredoxins or glutaredoxins on resistance to the superoxide anion (data not shown). These results indicate that both Trx1 and Trx2 can protect against oxidative stress induced by hydroperoxides. We therefore examined whether differences in oxidant sensitivity between the thioredoxin mutants might arise as a result of differential expression of TRX1 and TRX2.
Differential regulation of TRX1 and TRX2 expression
The expression levels of glutaredoxins and thioredoxins were previously found to be unaffected in mutants lacking combinations of glutaredoxins and thioredoxins, indicating that they are not regulated in a compensatory manner (Draculic et al., 2000). However, TRX2 expression is known to be 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; Lee et al., 1999). In contrast, little is known regarding the regulation of TRX1 expression and whether it is regulated in response to stress or growth conditions. We therefore used TRX1::lacZ and TRX2::lacZ reporter constructs to compare the regulation of TRX1 and TRX2 expression (Fig. 2A). The basal level of TRX2::lacZ expression was fourfold higher than the basal level of TRX1::lacZ expression. In addition, the expression of TRX2 was induced twofold in response to 0.2 mM H2O2 and sevenfold in response to 1.5 mM diamide. Stationary phase growth resulted in a large 20-fold increase in TRX2::lacZ expression. In contrast, TRX1 expression was unaffected by treatment with H2O2 or diamide, but a low sixfold increase in expression was seen upon entry into stationary phase. Thus, the apparent difference in sensitivity of the trx1 and trx2 mutants to H2O2 may result from the fact that TRX2 expression can be regulated in response to changing conditions. In particular, TRX2 expression is upregulated in response to ROS and stationary phase growth, conditions in which TRX1 expression remains largely unchanged.
The finding that thioredoxin expression is increased during stationary phase indicates that thioredoxin function may be particularly important during this phase of growth. Thus, we tested whether there are any growth phase-specific differences in oxidant sensitivity between the thioredoxin mutants. Specifically, wild-type and thioredoxin mutant cells were grown to exponential phase (A600 = 1) or stationary phase and tested for resistance to H2O2 (Fig. 2B). The wild-type and trx1 mutant strains showed similar levels of resistance during both growth phases. Double mutants lacking both TRX1 and TRX2 were sensitive to H2O2 during exponential and stationary phase growth, confirming the requirement for thioredoxins to detoxify hydroperoxides. In contrast, the trx2 mutant was sensitive to H2O2 during stationary phase growth, whereas it showed wild-type resistance during exponential phase growth. These results indicate that Trx1 and Trx2 are interchangeable as antioxidants during normal aerobic growth but, during stationary phase growth, TRX1 alone is insufficient to provide protection against H2O2.
Thioredoxins are essential for stationary phase survival
Given the stationary phase requirement for thioredoxin antioxidant function, we examined whether thioredoxins are required for stationary phase survival. The wild-type and thioredoxin mutants were grown into stationary phase, washed and resuspended in water as described in Experimental procedures. Cell viability was then moni-tored over time. The wild type, trx1 and trx2 mutants maintained 100% viability for over 30 days (Fig. 3). In contrast, the trx1 trx2 mutant started with a lower viability and died much sooner, losing >90% viability within 30 days. These data indicate a requirement for thioredoxins during stationary phase growth.
Role of thioredoxins as cofactors for thioredoxin peroxidases
As cells enter stationary phase, they undergo a complex series of physiological changes, which also result in an increased resistance to oxidative stress (Werner-Washburne et al., 1993; Jamieson et al., 1994). As a key antioxidant function of thioredoxins is their role as a cofactor for thioredoxin peroxidases, we determined whether any of the thioredoxin peroxidases are required for the stationary phase increase in oxidant resistance. The wild-type and isogenic mutant strains were grown to exponential phase (A600 = 1) or stationary phase and spotted onto plates containing various concentrations of H2O2 (Fig. 4A). The pattern of oxidant sensitivity seen in the mutants was in agreement with previously published data for strains lacking thioredoxin peroxidases (Park et al., 2000). Namely, the strain lacking TSA1 was the most sensitive (Fig. 4A, 10 mM H2O2), and strains lacking tsa2 (ahp1) and YDR453c were sensitive to higher concentrations of H2O2 (Fig. 4A, 11 mM H2O2). However, our data indicate that, with the exception of the tsa1 mutant, all the thioredoxin peroxidase mutants and the wild-type strain show increased resistance to H2O2 during stationary phase growth compared with exponential phase growth. In contrast, the tsa1 mutant is equally sensitive to H2O2 during both growth phases. These data indicate that Tsa1 is required for the increased resistance to H2O2 observed during stationary phase growth.
We next tested the role of Tsa1 in thioredoxin-mediated resistance to hydroperoxides. First, we tested whether overexpression of TRX1 could increase resistance to H2O2 during stationary phase growth in the strains lacking thioredoxin peroxidases (Fig. 4B). Multicopy TRX1 was found to increase H2O2 resistance in all the mutants tested, including the tsa1mutant strain. Secondly, we examined the role of Trx1 and Trx2 as cofactors for Tsa1. Overexpression of TSA1 in the wild-type and trx1 mutant strains was found to result in an increased resistance to 9 mM H2O2 (Fig. 4C). Although the trx2 mutant is hypersensitive to H2O2, overexpression of TSA1 was still found to increase resistance to H2O2 at lower concentrations (Fig. 4C, 6 mM). No increase in H2O2 resistance was observed in the trx1 trx2 mutant, indicating that thioredoxins are the physiological electron donors for Tsa1. Thus, both Trx1 and Trx2 are able to support the detoxification of H2O2 mediated by Tsa1, again confirming that the yeast thioredoxins are interchangeable as antioxidants. Interestingly, similar results were found with tert-butyl hydroperoxide, indicating that Tsa1 can also mediate the detoxification of an organic hydroperoxide using reducing power from thioredoxins (Fig. 4C). Thus, at least part of the stationary phase resistance to hydroperoxides is mediated by thioredoxins via the antioxidant activity of Tsa1.
Role of thioredoxins in the regulation of the cellular redox state
GSH is an abundant low-molecular-weight thiol that is readily oxidized by various antioxidant enzymes and free radicals and has been widely used as an indicator of the cellular redox state. Loss of thioredoxins has been shown previously to result in an increase in the levels of oxidized glutathione (GSSG; Muller, 1996). However, these studies were made using cells grown on rich YEPD media, which contains significant amounts of exogenous glutathione (see Discussion). We therefore examined the effects of thioredoxin mutants on glutathione metabolism in cells grown on minimal SD media to determine whether thioredoxins regulate glutathione redox state and metabolism in a growth phase-dependent manner.
Strains were grown to exponential or stationary phase and treated with H2O2 (4 mM per A600 of cells) for 1 h (Fig. 5). Exponential phase yeast cells contained ≈ 60-fold more GSH than GSSG, resulting in a redox ratio (GSH:GSSG) of 64. In response to H2O2, oxidized GSSG was increased approximately twofold, lowering the redox ratio to 22. Stationary phase yeast cells contained similar levels of GSH and GSSG to the exponential phase cells. However, in response to H2O2, there was no increase in GSSG, and the levels of GSH were somewhat increased during the treatment period, resulting in a redox ratio of 100. The finding that the glutathione redox ratio is maintained in response to H2O2 is in agreement with the fact that stationary phase yeast cells are more resistant to oxidative stress. No differences in the levels of GSH or GSSG were detected in the trx1 mutant during exponential or stationary phase growth, or after exposure to H2O2, compared with the wild-type strain. In the trx2 mutant, there were again no differences in the levels of GSH or GSSG during either growth phase. However, treatment of stationary phase cells with H2O2 increased the levels of GSSG by ≈ 50% and, hence, the GSH:GSSG ratio remained constant despite an increase in the levels of GSH. This decrease in the redox ratio correlates with the increased sensitivity of trx2 mutants to H2O2 during stationary phase growth (Fig. 2). In contrast to the wild type and single thioredoxin mutants, the trx1 trx2 mutant contained elevated levels of GSH, which were increased three- and 1.6-fold during exponential and stationary phase growth respectively. In addition, GSSG was elevated 15-fold during exponential phase growth, lowering the glutathione redox ratio to 14. In response to H2O2, GSSG was increased further to a level at which oxidized glutathione represented 12% of the total free glutathione in the cell. During stationary phase growth, the levels of GSSG were comparable with those in the wild strain, but increased fivefold in response to H2O2. These results indicate that thioredoxins are required to maintain the redox state of glutathione in a growth phase-dependent manner as well as in response to oxidative stress imposed by hydroperoxides.
During exposure to ROS, GSH can form mixed disulphides with proteins in a reaction termed glutathiolation (Grant et al., 1998; Klatt and Lamas, 2000). Protein-bound GSH (GSSP) was measured by acid precipitation of proteins followed by reduction with sodium borohydride (Fig. 5). Low levels of protein-bound glutathione were detected in a wild-type strain, comparable with the levels of glutathione present in the oxidized GSSG form. However, there was a large sevenfold increase in GSSP during stationary phase growth. This growth phase-dependent increase in GSSP was significantly higher than the 1.5-fold increase seen in exponential phase cells in response to H2O2. Surprisingly, the high levels of GSSP present in stationary phase cells were reduced by ≈ 40% in response to H2O2 treatment, indicating that the release of GSH bound to proteins may be required to protect against oxidative stress. The trx1 and trx2 mutant strains contained comparable levels of GSSP to the wild-type strain during both growth phases. In addition, the high levels of GSSP present during stationary phase growth were reduced after exposure to H2O2. In contrast, the trx1 trx2 mutant contained constitutively high levels of GSSP during both exponential and stationary phase growth. In response to H2O2, there was no decrease in GSSP in the stationary phase cells. Thus, the levels of protein-bound glutathione in the cell depend on the presence of thioredoxins and are regulated in a growth phase-dependant manner.
The thioredoxin system is required to regulate protein glutathiolation
To examine the requirement for thioredoxins to regulate GSSP levels, we measured their levels in a strain deleted for TRR1. Loss of thioredoxin reductase was found to result in a threefold increase in the levels of both GSSG and GSH, resulting in a wild-type redox ratio (Fig. 6). However, similar to the trx1 trx2 mutant, the levels of GSSP were elevated 10-fold in the trr1 mutant. The increase in GSSP was not simply a result of lacking an antioxidant, as GSSP levels were unaffected in a strain lacking cytosolic catalase (ctt1; Fig. 6). Similarly, the increase in GSSP did not arise as a consequence of the increase in GSSG levels, as GSSP levels were unaffected in a strain lacking glutathione reductase (glr1; Fig. 6) and, hence, containing high levels of GSSG. Finally, strains lacking glutaredoxins (grx1 grx2; Fig. 6), were unaffected in GSSP levels.
All aerobic organisms are exposed to 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. Left unchecked, these ROS can cause wide-ranging damage to macromolecules and are believed to have a causal relationship with many disease processes (Halliwell and Gutteridge, 1989; Gutteridge, 1993). To protect against oxidant damage, cells contain effective antioxidant defence systems, and these have been studied extensively using the yeast Saccharomyces cerevisiae (Jamieson, 1998). Recent reports have highlighted the key role played by sulphydryl groups (–SH) in the response to oxidative stress and, in particular, the roles of the GSH/glutaredoxin and thioredoxin systems in maintaining the redox homeostasis of the cell (Carmel-Harel and Storz, 2000). Yeast contains cytoplasmic thioredoxins and glutaredoxins as well as a mitochondrial thioredoxin system (Trx3) and three glutaredoxin-related (Grx3–5) enzymes (reviewed by Grant, 2001). The presence of these overlapping systems begs the question as to why yeast maintains this apparent redundancy. Redundancy and functional overlap has been seen for a number of antioxidant defence systems. For example, yeast contains two catalases and two superoxide dismutases, located in different cellular compartments, but with overlapping functions (Izawa et al., 1996; Longo et al., 1996; Flattery-O’Brien et al., 1997). Redundancy in such important protective systems may provide an evolutionary advantage. In the present study, we have investigated the apparent redundancy in the antioxidant function of the cytoplasmic thioredoxins and glutaredoxins.
The availability of triple mutants (Draculic et al., 2000) has enabled us to determine the roles of thioredoxins and glutaredoxins individually in the cell. Thioredoxins were found to be more important than glutaredoxins in mediating protection against hydroperoxides, with the presence of TRX2 required for wild-type levels of resistance. In stationary phase cells, the presence of TRX2, but not TRX1 alone, was sufficient to confer resistance to both H2O2 and t-BH. This is the first indication that Trx1 and Trx2 might have different functions in the cell. In all previous studies, the cytoplasmic thioredoxins appeared to play redundant roles. For example, growth rate, sulphate assimilation and activation of the transcription factor yAP-1 are all defective in trx1 trx2 mutants, but are maintained at wild-type levels in either the trx1 or the trx2 single mutants (Muller, 1991; Izawa et al., 1999). This is perhaps not surprising, given that Trx1 and Trx2 are highly homologous (74% identity; Muller, 1991). Similarly, the results presented in the current study indicate that differences in oxidant sensitivity may actually arise because of differences in gene expression rather than biochemical activity. Three lines of evidence suggested that the thioredoxins are redundant for their activity as antioxidants. First, overexpression of either TRX1 or TRX2 leads to an increased resistance to hydroperoxides. Secondly, Trx1 or Trx2 are both able to act as cofactors for thioredoxin peroxidase (Tsa1)-mediated resistance to hydroperoxides. Thirdly, the sensitivity of the trx2 mutant to hydroperoxides was not observed during normal aerobic growth and was only found during stationary phase growth. Furthermore, gene expression analysis showed that this was a growth condition in which TRX2 and, to a lesser extent, TRX1 expression was upregulated. Thus, Trx1 and Trx2 appear to be functionally redundant as antioxidants. The reason that S. cerevisiae maintains two genes, with the associated energetic costs, is probably related to their differential regulation. Expression of TRX2, in particular, can be upregulated in response to changing growth conditions, providing a first line of defence against oxidative stress. TRX1 may serve an ancillary or back-up role during conditions in which TRX2 is insufficient to provide an antioxidant defence.
Yeast cells grown on glucose-based media enter stationary phase after the exhaustion of glucose from the media (reviewed by Werner-Washburne et al., 1993; 1996). As cells enter stationary phase, they undergo a complex series of physiological changes and become resistant to various stress conditions including heat and oxidative stress (Jamieson, 1992; Werner-Washburne et al., 1993; Jamieson et al., 1994). Stationary phase survival of yeast cells is increasingly becoming used as a model of chronological lifespan in ageing studies (Gershon and Gershon, 2000). Such studies have implicated the key role of oxidative stress in ageing (Jakubowski et al., 2000) and the requirement for antioxi-dants such as superoxide dismutase (Longo et al., 1996). In this study, we have shown that thioredoxins are also required for stationary phase survival in yeast cells. Either Trx1 or Trx2 alone was sufficient to ensure wild-type longevity, but loss of both thioredoxins resulted in a rapid loss of viability. This stationary phase requirement was found to depend on the activity of thioredoxins both as a cofactor for thioredoxin peroxidase (Tsa1) and also to maintain the cellular redox state.
Similar to mammalian cells, a large number of thioredoxin peroxidase isoforms have been identified from the yeast genome sequencing project (Park et al., 2000). In the present study, we found that both Trx1 and Trx2 can act as cofactors for Tsa1, and that Tsa1 is essential for the increased resistance to hydroperoxides observed upon entry of yeast cells into stationary phase. The functions of the other thioredoxin peroxidase isoenzymes are as yet unknown, but they appear to be unable to sub-stitute for the thioredoxin-mediated antioxidant activity of Tsa1. However, overexpression of TRX1 in a tsa1 mutant could still increase resistance to H2O2, indicating that the antioxidant activity of thioredoxins does not depend on Tsa1 alone.
Previous studies have shown that exponential phase yeast cells grown on rich YEPD media have a redox ratio (GSH:GSSG) of ≈ 11–16:1, indicating that most of the intracellular glutathione is maintained in a reduced (GSH) form (Grant et al., 1996; Muller, 1996). Loss of both TRX1 and TRX2 resulted in an increase in the levels of GSSG, indicating a functional link between the thioredoxin system and the redox status of GSH in the cell (Muller, 1996). However, these studies were conducted using cells grown on rich YEPD media, which contains yeast extract and, hence, significant amounts of exogenous glutathione largely present in an oxidized form. We therefore examined glutathione metabolism in the thioredoxin mutants grown on minimal media during different growth phases. The redox ratio in normal aerobic cells was un-affected by loss of TRX1 or TRX2 alone, confirming the redundant nature of Trx1 and Trx2 as antioxidants. However, the loss of both TRX1 and TRX2 shifted the redox ratio to 14 as a result of an increase in the levels of both GSSG and, to a lesser extent, GSH. Surprisingly, the redox ratio in the trx1 trx2 mutant was restored to wild-type levels in stationary phase cells on account of a reduction in the levels of GSSG. This may result from the known stationary phase induction of various other anti-oxidants, including catalase (CTT1), superoxide dismutase (SOD2), glutathione reductase (GLR1) and glutaredoxins (GRX1, GRX2) (Schuller et al., 1994; Grant et al., 1996; 2000; Flattery-O’Brien et al., 1997). However, stationary cells lacking TRX1 and TRX2 were unable to maintain GSSG levels in response to a challenge with H2O2. Thus, thioredoxins are required to regulate the redox state and levels of glutathione in response to oxidants in a growth phase-dependent manner.
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; Klatt and Lamas, 2000). Protein S-thiolation may also represent a novel form of post-translational modification that can regulate protein activity in response to growth or other cellular signals. In agreement with this suggestion, S-thiolation is reversible, and dethiolation can be catalysed in vitro by glutare-doxins and thioredoxins (Thomas et al., 1995). However, in vitro dethiolation can also be achieved using low-molecular-weight thiols such as GSH or cysteine, making the identity of the physiological reductants unclear. In the present study, we have demonstrated for the first time that the levels of GSSP in the cell are dependent on the presence of thioredoxins, and are constitutively high in a trx1 trx2 mutant. Wild-type cells contained low levels of GSSP during exponential phase, which did not increase significantly in response to oxidative stress. In contrast, GSSP formation was dependent on the growth phase and was elevated sevenfold upon entry into stationary phase. Interestingly, these high levels of GSSP were reduced in response to treatment with H2O2. This decrease in GSSP was also dependent on the presence of both TRX1 and TRX2 and may indicate that the mobilization of glutathione from a protein-bound to a free form can serve an antioxidant function.
In summary, we have shown that Trx1 and Trx2 have redundant functions as antioxidants, and that their activity is particularly required during stationary phase growth. Further studies of thioredoxins and glutaredoxins in yeast will allow for a more complete understanding of these key peptides in eukaryotes and, in particular, their roles in both normal and pathological cellular processes.
Yeast strains and growth conditions
The S. cerevisiae strains used in this study are described in Table 1. Strains were grown in rich YEPD medium (2% w/v glucose, 2% w/v bactopeptone, 1% w/v yeast extract) or minimal SD media (0.17% w/v yeast nitrogen base without amino acids, 5% w/v ammonium sulphate, 2% w/v glucose) supplemented with appropriate amino acids and bases: 2 mM leucine, 4 mM isoleucine, 1 mM valine, 0.3 mM histidine, 0.4 mM tryptophan, 1 mM lysine, 0.15 mM adenine and 0.2 mM uracil. Media were solidified by the addition of 2% (w/v) agar. Sensitivity to oxidants was determined by growing cells to exponential phase (A600 = 1) or stationary phase (48 h growth) and spotting onto agar plates containing various concentrations of H2O2 or tert-butylhydroperoxide. Growth to an A600 of 1 in minimal SD media was defined as exponential phase growth based on our previous growth curve experiments for the various glutaredoxin and thioredoxin mutants (Draculic et al., 2000). Similarly, stationary phase was defined as 48 h growth in minimal SD media as, at this time point, the cells had passed through the diauxic phase of growth, and there was no longer any change in cell density (Draculic et al., 2000).
Table 1. Yeast strains used in this study.
The genes deleted in these strains have not yet been given standard yeast genetic names.
MATa ura3-52 leu2-3,112 trp1-1 ade2-1 his3-11 can1-100
Stationary phase survival was determined by first growing cells into stationary phase in 40 ml of SD media in 250 ml flasks. Cells were washed twice in sterile distilled water before resuspending in a final volume of 40 ml of water. Cultures were incubated at 30°C with shaking, and viable counts were determined by diluting aliquots of cells into YEPD medium and plating in triplicate on YEPD plates.
Multicopy plasmids containing GRX1 (pG501) and GRX2 (pL4) have been described previously (Luikenhuis et al., 1997). The TRX1, TRX2 and TSA1 genes were isolated by polymerase chain reaction (PCR) amplification of total yeast DNA with oligonucleotides specific for TRX sequences. For TRX1, a 1750 bp fragment was amplified using oligonucleotides that hybridized 770 bp upstream of the putative ATG start codon (5′-TTCCCAAACAACACCTACGGATCCTCTCAAGTCTTATGGG-3′) and 670 bp downstream of the TAA stop codon (5′-GTGGCCAGGTGCTTCTTGTTCAGAAC-3′) respectively. TRX1 was cloned into the polylinker region of plasmid pRS426 (Christianson et al., 1992) using a BamHI restriction site introduced by the 5′ oligonucleotide (underlined) and a naturally occurring EcoRI site. For TRX2, a 2074 bp fragment was amplified using oligonucleotides that hybridized 786 bp upstream of the putative ATG start codon (5′-GAAATTTCCAACTGTGTTGGATCCCAACAAGATTGACCA-3′) and 895 bp downstream of the TAG stop codon (5′-ACTATCGGCCAGAGCTACCGATATAT-3′) respectively. The amplicon was cloned into the polylinker region of plasmid pRS424 (Christianson et al., 1992) using a BamHI restriction site introduced by the 5′ oligonucleotide (underlined) and a naturally occurring ClaI site. For TSA1, a 1403 bp fragment was amplified using oligonucleotides that hybridized 485 bp upstream of the putative ATG start codon (5′-TTCCAGACCCGGAATTCGATAGGGT-3′) and 334 bp downstream of the TAA stop codon (5′-TTGACGCCCTTCCCAAAGAGGAAAC-3′) respectively. The amplicon was cloned into the polylinker region of plasmid pRS423 (Christianson et al., 1992) using an EcoRI restriction site introduced by the 5′ oligonucleotide (underlined) and a naturally occurring EcoRI site.
The TRX2::lacZ plasmid was a kind gift from S. Moye-Rowley (Iowa) and contained a 500 bp EcoRI–BamHI promoter fragment from TRX2 fused in frame with the lacZ gene. The TRX1::lacZ plasmid was constructed by PCR to replace the TRX2 promoter fragment with an equivalent 500 bp EcoRI–BamHI promoter fragment from TRX1.
Determination of glutathione levels
Glutathione levels were determined as described previously (Grant et al., 1998). Briefly, cells were grown in minimal SD medium to an A600 of 1 (1–2 × 107 cells ml–1) or into stationary phase and harvested by centrifugation. Cells were washed twice with phosphate-buffered saline (PBS; pH 7.4) to remove any traces of growth medium and resuspended in ice-cold 8 mM HCl, 1.3% (w/v) 5-sulphosalicyclic acid. Cells were broken with glass beads using a Minibead beater (Biospec Scientific) for 30 s at 4°C, before incubation on ice for 15 min to precipitate proteins. Cell debris and proteins were pelleted in a microcentrifuge for 15 min (13 000 r.p.m. at 4°C), and the supernatant was used for the determination of free glutathione. To release protein-bound glutathione (GSSP), the pellets from the sulphosalicyclic acid extraction were treated with 1% sodium borohydride. GSH levels are expressed as nmol of GSH per A600 of cells.
For the determination of β-galactosidase activity, transformants were assayed essentially as described previously (Rose and Botstein, 1983). Cells were grown to early exponential phase (A600 = 1) at 30°C before treatment with H2O2 (0.2 mM) or diamide (1.5 mM) for 1 h. For stationary phase analysis, cells were grown for 48 h as described above. β-Galactosidase activity is expressed as nmol of ONPG hydrolysed min–1μg–1 total protein (U). Values shown are the means of at least three independent determinations.
This work was supported by project grant no. 36/G13234 from the Biotechnology and Biological Sciences Research Council (BBSRC). The trr1 mutant was a kind gift from Garry Merril, and the trx2::lacZ fusion was from W. Scott Moye-Rowley. We thank Kathryn Quinn for critical reading of the manuscript.