Coupling of the Transcriptional Regulation of Glutathione Biosynthesis to the Availability of Glutathione and Methionine via the Met4 and Yap1 Transcription Factors*

Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest. Here, we show that the Yap1 and Met4 transcription factors regulate the expression of γ-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion. Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered. Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response. The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1. In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion. Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation. These results indicate that the Yap1-dependant activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependant mechanism.

The ability to regulate biosynthetic pathways is a fundamental aspect of adaptation to life in a changing environment. In response to a stress, cells must be able to prioritize the allocation of resources to support the increased demand for defensive mechanisms. Understanding the mechanisms that facilitate stress-induced changes is important in determining how a cell co-ordinates different areas of metabolism.
An inducible stress response has been described for the bio-synthesis of glutathione (GSH) in the yeast Saccharomyces cerevisiae. GSH is a tripeptide (␥-L-glutamyl-L-cysteinylglycine) that plays an important role in protecting yeast cells against damage induced by oxidative stress (1)(2)(3). GSH counters the potentially damaging effect of reactive oxygen species through direct scavenging of free radicals and through the action of antioxidant enzymes such as the glutathione peroxidases (4). In addition GSH has other protective roles within the cell, such as the detoxification of xenobiotics and heavy metals through the formation of GSH conjugates and their subsequent export into the vacuole (5). The reversible binding of GSH to protein sulfhydryl groups can protect them from irreversible oxidative damage (6,7). There are also requirements for GSH in methylglyoxal detoxification, as a cofactor for ribonucleotide reductase, in protein folding, and in amino acid transport (4,8). The first step in the synthesis of GSH is the conjugation of glutamate and cysteine by ␥-glutamylcysteine synthetase, encoded by GSH1 (9,10). This produces ␥-glutamylcysteine, to which glycine is added by glutathione synthetase, encoded by GSH2 (11,12). Mutant strains lacking GSH1 are unable to grow in the absence of GSH, indicating that this metabolite is essential in S. cerevisiae (1,13,14). The rate-limiting step in the biosynthetic pathway is Gsh1, which is feedback-inhibited at the enzyme level by GSH (4). The rate of GSH1 expression is, therefore, very important in regulating the abundance of Gsh1 and determining the rate of GSH biosynthesis. In response to oxidative stress, GSH1 expression is increased in a Yap1-dependant manner (15)(16)(17). Yap1 is a redox-sensitive bZip-transcription factor that regulates the expression of many antioxidant genes (18 -20). Upon exposure to H 2 O 2 , Yap1 accumulates in the nucleus due to the masking of a C-terminal nuclear export signal by an intramolecular disulfide bond (21,22). It has recently been shown that the glutathione peroxidase likeenzyme, Gpx3, is required for the H 2 O 2 -dependent formation of the intramolecular disulfide bond (23). The pathway is turned off by thioredoxin, which reduces both Gpx3 and Yap1. There is a requirement for Yap1 in the response of GSH1 to oxidants such as H 2 O 2 , tert-butyl hydroperoxide, and menadione and also to other stresses such as heat shock and cadmium (16,24,25). Deleting the Yap1-responsive element (YRE) 1 in the GSH1 promoter still permits H 2 O 2 -mediated induction, which may indicate that Yap1 only plays an indirect role in the response to H 2 O 2 (17).
Cadmium-inducible expression of GSH1 also requires the presence of the Met4 transcription factor (26). Met4 is a bZip-transcriptional activator that regulates the assimilation of extracellular sulfate into the sulfur-containing amino acids methionine and cysteine (27). Met4 will, therefore, have an indirect effect on GSH biosynthesis by regulating the supply of cysteine. Met4 lacks a DNA binding domain and is tethered to DNA in the form of a heteromeric complex involving the bZip protein, Met28, and the basic helix-loop-helix protein, Cbf1 (28,29). Met4 may also be recruited to DNA in an alternative complex involving Met28 and one of the zinc finger proteins, Met31 or Met32 (30,31). The Met4-regulated genes of the sulfate assimilation pathway have differing requirements for one or both of these complexes. The GSH1 promoter contains binding motifs for both Cbf1 and Met31/32 (26). The sulfate assimilation pathway is turned off upon exposure to methionine, which triggers the ubiquination of Met4 (32,33). The nature of this regulation is complex because ubiquitination inhibits Met4 transcriptional activity through altered promoter recruitment or through Met4 degradation depending upon the media composition and environment of the individual promoters (34).
In this work we investigated how GSH biosynthesis is regulated in response to depletion of the GSH pool. We have previously determined that there is Met4-dependant induction of GSH1 expression after GSH depletion (35). Here, we identify a role for the stress-responsive transcription factor Yap1 in this response and propose a mechanism in which these two transcription factors combine to regulate GSH1 expression. In addition, we demonstrate that the regulation of GSH1 expression by this mechanism is important in defense against xenobiotics, which deplete cellular GSH concentrations.
Determination of Glutathione and Thioredoxin Redox States-Concentrations of free (GSH and GSSG) and protein-bound (GSSP) glutathione were determined as described previously (37). The redox state of thioredoxins was measured by covalent modification with the thiolreactive probe 4-acetamido-4Јmaleimidyldystilbene-2,2Ј-disulfonic acid (Molecular Probes) as described previously (38).
␤-Galactosidase Assays-For the determination of ␤-galactosidase activity, transformants were assayed essentially as described previously (40). Activity is expressed as nmol of o-nitrophenyl-␤-D-galacto-pyranoside hydrolyzed/min/g of total protein (units). All ␤-galactosidase experiments were repeated at least twice, and a representative plot is shown. Values shown are the means of at least two independent determinations. Error bars denote S.E.
Microarray Hybridizations and Data Analysis-Yeast cells were grown in triplicate to mid-exponential phase in minimal SD media. Growth conditions for the gsh1 mutant were predetermined to allow maximal GSH depletion in mid-exponential phase without a decrease in growth rate. Total depletion of the GSH pool in the gsh1 mutant leads to a cell cycle arrest. To examine the transcriptome in GSH-depleted cells that were still growing normally, cultures of the gsh1 mutant were inoculated with the lowest volume of a GSH-containing stationary phase culture that still allowed a normal growth rate to an A 600 of 0.5-0.6. Preparation of RNA, probes, and hybridization to whole yeast genome microarrays (YG-S98, Affymetrix) was performed as described on the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) web site (www.cogeme.man.ac.uk). Data acquisition was performed using Affymetrix Microarray Suite Version 5.0 software and analyzed using dChip v1.1 software (41). The mean expression values from three independently grown yeast cultures were used for comparative analysis. Gene expression was deemed to be significantly different between strains if it fulfilled the following criteria; 1) the fold change of gene expression was greater than 2 at the 90% confidence level, 2) the mean expression values are significantly different using an unpaired t test (p Ͻ 0.05), 3) the mean expression values differ by greater than 50, 4) the gene is called present in greater than 60% of the arrays. For the comparison to other microarray data sets, data from the YAP1 overexpression and trr1 mutant analyses were downloaded from Stanford Genomic Resources web site (genome-www.stanford.edu/yeast_stress and genome-www.stanford.edu/trr1, respectively) (19,20). Genes exhibiting a log 2 expression ratio higher than 1.0 were compared with the data set obtained in the gsh1 mutant. The identification of prospective regulatory sequences within promoters was performed by RSAT (Regulatory Sequence Analysis Tools, rsat.ulb.ac.be/rsat).

The Regulation of the Sulfate Assimilation Pathway Is Not
Affected by Cellular GSH Concentrations-The regulation of metabolic pathways is essential for cell adaptation to differing environmental conditions. In this paper we have investigated the mechanisms regulating the biosynthesis of the key antioxidant, GSH. Our previous work indicated that expression of GSH1 is strongly induced in response to GSH depletion and that this response is dependent on the presence of the Met4 transcription factor (35). To understand the mechanisms regulating GSH1 expression, we were particularly interested in the transcriptional responses of other Met4-dependent genes in response to GSH depletion. We, therefore, analyzed the global transcriptional response to GSH depletion in a gsh1 mutant. Microarray analysis revealed that there is a significant effect on the transcriptome, with 151 open reading frames significantly up-regulated and 38 down-regulated. Here we will only refer to the findings that are relevant to the regulation of GSH1 expression, since a detailed analysis of these data is beyond the scope of this paper and will be described elsewhere. 2 The microarray analysis of the gsh1 mutant indicates that lower cellular GSH concentrations do not lead to a general induction of the sulfate assimilation pathway. There is no significant effect on the expression of genes involved in sulfur metabolism other than a 3.3-fold increase in STR3 transcripts. The STR3 gene encodes cystathionine ␤-lyase, which catalyzes the conversion of cysteine to cystathionine and would, thus, prevent a toxic accumulation of cysteine in the gsh1 mutant (42).
To confirm the effect of GSH depletion on the regulation of the sulfate assimilation pathway, we analyzed the transcriptional response of MET3, MET16, and MET25 using Northern blot analysis (Fig. 1A). These genes have previously been shown to be controlled by the Met4 transcriptional activator protein (27). However, none of these genes was induced by GSH depletion in the gsh1 mutant nor were they repressed by the exogenous addition of 1 mM GSH. This is in strong contrast with the induction of GSH1 expression observed after GSH depletion (Fig. 1B). The addition of 1 mM methionine severely repressed the expression of all three sulfate assimilation genes and also repressed the GSH1 induction observed in the gsh1 mutant. Thus, although the expression of the GSH1 gene is regulated in response to GSH availability, the expression of other known Met4 target genes is unaffected under these con-ditions. This indicates that Met4 acts to specifically up-regulate GSH biosynthesis in response to GSH depletion. We, therefore, examined the microarray data generated from the gsh1 mutant for evidence that other transcription factors may contribute to this specificity.
GSH Depletion Activates Yap1-regulated Genes-Examination of the microarray data revealed that a number of genes that are regulated by the Yap1 transcriptional activator protein are up-regulated in the gsh1 mutant (Table I). For example, mutant strains of yeast lacking thioredoxin reductase (trr1) exhibit a constitutive Yap1 response, indicating that the thioredoxin system is integral for the redox regulation of this transcription factor (19). Comparison of the whole-genome expression data identified 16 genes in the gsh1 mutant from a set of 35 genes in the trr1 mutant that are strongly induced (greater than 2-fold) in both mutants. The group of genes up-regulated in a gsh1 mutant also contains 25 genes that have previously been shown to be up-regulated (from a set of 118 genes) after the overexpression of plasmid-borne YAP1 in wildtype cells (20). Furthermore, analysis of the promoter regions of the genes up-regulated in the gsh1 mutant indicates that a significant proportion (43 of 151) contain a putative Yap1 binding site (TTAC/GTAA or TGACTAA).
To determine whether Yap1 oxidation in response to GSH depletion was due to an altered redox status, we performed microarray analysis on the glr1 mutant. Mutant strains lacking GLR1, encoding glutathione reductase, are unable to recycle oxidized GSSG back to GSH, and consequently, the redox status of the GSH pool is much more oxidized (36). However, oxidation of the GSH pool does not result in Yap1 activation because no target genes were up-regulated in the glr1 mutant (data not shown).
Given the elevated expression of Yap1 targets genes in the gsh1 mutant, we confirmed that Yap1 is activated in a gsh1 mutant using a YRE::lacZ reporter construct. This construct contains three YRE from the promoter of GSH1 fused upstream of the ␤-galactosidase reporter gene (15). The gsh1 mutant strain exhibited a 5-fold increase in YRE::lacZ expression compared with the wild-type control (data not shown). These data indicate that GSH depletion leads to an activation of the Yap1 transcription factor, which has previously been shown to regulate GSH1 expression in response to oxidative stress (43). We, therefore, confirmed that Yap1 also regulates GSH1 expression after GSH depletion using a gsh1 yap1 double mutant. Deletion of YAP1 in a gsh1 mutant reduced the increase in GSH1 expression ( Fig. 2A). In addition, mutation of the Yap1-responsive element in the GSH1 promoter prevented the GSH-mediated induction of expression, confirming the requirement for Yap1 ( Fig. 2A). Thus, both Met4 and Yap1 are required for elevated GSH1 expression after GSH depletion.
Loss of CBF1 Removes the Requirement for Met4 in GSH Biosynthesis-To further examine the requirements for Met4 and Yap1 we investigated the role of Cbf1 in GSH biosynthesis. Met4 contains no DNA binding domain and interacts with promoters through a complex containing Met28 and either Cbf1 or Met31/32 (28,31). As a consequence, many genes of the sulfate assimilation pathway are poorly expressed in a cbf1 mutant, leading to methionine auxotrophy (44,45). In contrast, the expression of a GSH1::lacZ reporter is constitutively elevated in a cbf1 mutant, suggesting a repressive role for this protein in regulating GSH1 expression (26). We, therefore, constructed a series of mutants in which the CBF1 gene was deleted in combination with either MET4 or YAP1 (Fig. 2B). Our data demonstrate that the induction of GSH1 expression in response to GSH depletion is still observed in the cbf1 mutant. This indicates that the interaction between Cbf1 and FIG. 1. The sulfate assimilation pathway is not regulated by GSH availability. A, Northern blot analysis of MET16, MET25, and MET3 was performed on exponential phase wild-type and gsh1 mutant cells grown in SD media in the presence of 1 mM methionine (Met), 1 mM GSH, or no addition (NA). ACT1 is shown as a loading control. B, GSH1 expression was determined using a GSH1::lacZ fusion in the wild-type (wt) and gsh1 mutant strains. ␤-Galactosidase activity was determined for exponential phase cultures grown in minimal media with no addition (control), 1 mM GSH, or 1 mM methionine.
Met4 is not required for the increase in GSH1 expression. The response to GSH depletion is even more pronounced in a met4 cbf1 gsh1 triple mutant, confirming that the requirement for Met4 is lost in a cbf1 background. A further indication that Met4 requires Cbf1 to regulate GSH1 expression was demonstrated by the inability of 1 mM methionine to prevent the induction of GSH1 expression in response to GSH depletion in the cbf1 mutant (Fig. 2B). In contrast, deleting YAP1 restored the basal levels of GSH1 expression in a cbf1 mutant and abrogated the induction of GSH1 expression normally seen in response to GSH depletion. This indicates that Yap1 is integral to the GSH depletion response regardless of the presence of Cbf1 and implies that Cbf1 normally acts to prevent the Yap1dependent transcriptional activation of GSH1.
GSH Depletion Induced by Exposure to CDNB-We have demonstrated that GSH biosynthesis in yeast is regulated in response to GSH depletion by increased expression of GSH1. This response was initially identified in a gsh1 mutant, which is unable to synthesize GSH. We wished to confirm that a similar response occurs in wild-type cells, which have been depleted of GSH to ensure that the response is not specific to the gsh1 mutant. The GSH pool in wild-type cells may be depleted after exposure to xenobiotics such as CDNB, which are rapidly detoxified by GSH conjugation (46). Thus, the re- sponse to xenobiotic exposure is a closer representation of the physiological situations that yeast may encounter in their natural environment. We first examined the effects of CDNB on the cellular GSH pool and the requirements for Met4 and Cbf1 in maintaining GSH concentrations. Total GSH levels (both free and bound to proteins) are lower in a met4 mutant compared with wild-type cells, indicating that Met4 is important in maintaining the GSH pool during normal aerobic growth ( Table II). An increase in total GSH levels was observed in both the cbf1 and met4 cbf1 mutants, consistent with their elevated basal expression of GSH1 (Fig. 2B). The addition of 0.08 mM CDNB for 2 h severely depleted total GSH concentrations in all strains, although the cbf1 and met4 cbf1 mutant strains still retained a significant concentration of cellular GSH. Importantly, CDNB did not increase GSH oxidation to GSSG in either the wild-type or met4 strains, confirming that CDNB selectively depletes the reduced GSH pool. However, there was a significant increase in GSSG in both cbf1 and met4 cbf1, suggesting CDNB increases GSH oxidation in these mutants.

Met4 and Yap1 Regulate GSH Biosynthesis in Response to
Exposure to CDNB-The results described above indicate that CDNB treatment effectively depletes the pool of GSH in yeast cells. We, therefore, investigated whether the transcriptional response to GSH depletion by CDNB mirrors which was caused by deletion of the GSH1 gene. Exposure to 0.08 mM CDNB for 2 h resulted in an increased expression of GSH1::lacZ (Fig. 3A). This response was dependent on both the Yap1 and Met4 transcription factors because GSH1 induction did not occur in either mutant. The requirement for Met4 was lost in a cbf1 background, confirming that Met4 is only required for the transcriptional response to GSH depletion in the presence of Cbf1. The regulation of GSH1 expression after GSH depletion induced by exposure to CDNB is, therefore, very similar to that seen in the gsh1 mutant.
GSH conjugation is very important in countering the toxicity of CDNB. Given that the met4 mutant is unable to increase the transcription of GSH1 after CDNB exposure, we examined whether this response is required in protecting yeast cells. Strains lacking MET4 were very sensitive to growth on CDNB compared with wild-type cells (Fig. 3B). However, a met4 cbf1 double mutant in which GSH1 expression is responsive to CDNB was not sensitive. These data indicate that the ability to up-regulate GSH biosynthesis is necessary for resistance to GSH-depleting agents.
Exposure to CDNB Causes Thioredoxin Oxidation and Yap1 Activation-Given that GSH depletion in a gsh1 mutant causes Yap1 activation, we tested whether exposure to CDNB has a similar effect. Expression of the YRE::lacZ reporter construct was elevated 8-fold upon CDNB treatment, indicating that Yap1 is strongly activated after exposure to this xenobiotic (Fig. 4A). In addition, CDNB exposure did not increase GSH1 expression in a construct containing a mutated Yap1-responsive element, confirming that Yap1 directly regulates GSH1 transcription in response to CDNB. Thus, GSH depletion, brought about by deletion of GSH1 or through exposure to a xenobiotic, activates Yap1, which induces the expression of GSH1 to increase GSH biosynthesis.
GSH is the major redox buffer in yeast, and its depletion FIG. 2. Yap1 and Met4 are required to regulate GSH1 expression in response to GSH depletion. A, expression of the GSH1::lacZ reporter construct was measured in wild-type (wt) and gsh1 mutant strains that had been deleted for MET4 or YAP1. The expression of a GSH1::lacZ reporter construct lacking the YRE was determined in wild-type and gsh1 strains. U, units. B, GSH1 expression was measured in wild-type and gsh1 mutant strains that had been deleted for CBF1, MET4, and CBF1 or YAP1 and CBF1. GSH1 expression was also determined in wild-type and gsh1 strains that had been deleted for CBF1 grown in the presence of 1 mM methionine.
would be expected to influence other redox systems such as the thioredoxin system. We have previously shown that a severe starvation for GSH, brought about by incubating the gsh1 mutant in media lacking GSH, results in oxidation of thioredoxins (Trx1 and Trx2) (39). We therefore examined whether the more physiological GSH depletion that occurs after exposure to CDNB could also affect the redox status of thioredoxins. Thioredoxin are predominately present in the reduced form in wild-type cells but are shifted to a more oxidized form after treatment with CDNB (Fig. 4B). In contrast, treatment with the thiol oxidizing agent diamide did not cause any thioredoxin oxidation. Diamide readily oxidizes glutathione but does not affect the total cellular concentrations of GSH (47), indicating that the oxidation of thioredoxins is specific for GSH depletion rather than any change in the glutathione redox state. Given that thioredoxins are required for Yap1 deactivation (22), we propose that thioredoxin oxidation resulting from GSH depletion is responsible for the activation of Yap1 and leads to the increased biosynthesis of GSH.
Met4 Couples Yap1-mediated GSH1 Expression to the Availability of Methionine-The co-regulation of GSH1 by both Yap1 and Met4 in response to GSH depletion suggests that there is a need to balance GSH biosynthesis with the sulfur requirements of the cell. From the data presented above, Yap1 appears to play a direct role as the transcriptional activator of GSH1 expression, and Met4 most likely plays a secondary regulatory role. We, therefore, attempted to determine whether Met4 acts to couple GSH biosynthesis to the cell demand for sulfate assimilation. The addition of methionine strongly represses the transcription of genes required for sulfate assimilation through a Strains were either untreated or treated with 0.08 mM CDNB for 2 h. Concentrations are shown for reduced (GSH), oxidized (GSSG), and protein-bound (GSSP) glutathione.

FIG. 3. Met4 and Yap1 regulate GSH biosynthesis in response to GSH-depletion induced by exposure to CDNB.
A, expression of the GSH1::lacZ reporter construct was measured in wild-type (wt), yap1, met4, cbf1, and met4 cbf1 strains after treatment with 0.08 mM CDNB for 2 h. B, sensitivity to CDNB was determined by spotting strains onto SD plates containing 0.02 or 0.03 mM CDNB. Cultures of wild-type, met4c cbf1, and met4 cbf1 strains were grown into stationary phase and adjusted to A 600 1.0, 0.1, and 0.01 before spotting onto plates. Growth was monitored after 3 days. U, units.
the inactivation of Met4. Similarly, methionine addition was found to abrogate the normal induction of GSH1 expression induced by CDNB treatment (Fig. 5, left panel). This effect was specific to GSH1 expression since the addition of methionine did not affect the induction of a YRE::lacZ reporter construct in response to CDNB (Fig. 5, right panel). Thus, CDNB causes Yap1 activation regardless of the presence of methionine but only induces GSH biosynthesis when methionine is absent. DISCUSSION Glutathione is an abundant and ubiquitous low molecular weight tripeptide whose biological importance is dependent upon its redox-active free sulfhydryl moiety. Many roles have been proposed for GSH in a variety of cellular processes including amino acid transport, synthesis of nucleic acids and proteins, modulation of enzyme activity, and metabolism of carcinogens, xenobiotics, and reactive oxygen species (4, 48 -51). Not surprisingly, therefore, GSH is an essential metabolite in eukaryotes. For example, mice that are deficient in GSH due to a targeted disruption of the gene encoding the first step in its synthesis die rapidly (52), and drug-induced GSH depletion results in many tissue pathologies including hemolysis and defective brain function, cataract formation, and oxidative damage to renal, hepatic, and brain tissues (53)(54)(55)(56). Similarly, loss of GSH from yeast results in a G 1 phase cell cycle arrest followed by a loss of viability after 3-4 days (14). We examined the transcriptional regulation of the GSH1 gene to identify the mechanisms to which cells have evolved to prevent such GSH depletion. GSH1 expression was found to be increased in response to GSH depletion in a response that is co-regulated by the Yap1 and Met4 transcription factors.
Previous reports indicate that Yap1 activity is regulated by the thioredoxin but not the glutathione redox state (19,22,57). Our data are the first to show that Yap1 activity is regulated by the GSH system. We have previously shown that starvation for GSH results in oxidation of thioredoxins (39), and in this present study we show that GSH depletion induced by exposure to CDNB has a similar effect. This may account for the activation of Yap1 because Yap1 is normally deactivated via thioredoxinmediated reduction of an intramolecular disulfide bond (22). Oxidation of the GSH pool in the glr1 mutant did not cause any activation of Yap1 target genes, in agreement with the observation that thioredoxin redox status is unaltered in a glr1 mutant (39). Thus, the Yap1-mediated increase in GSH1 expression only occurs when GSH depletion leads to thioredoxin oxidation. Although this would appear to be a relatively coarse control for regulating GSH homeostasis, GSH biosynthesis is also tightly regulated by feedback inhibition at the enzyme level. Thus, a minor decrease in the GSH pool would lessen feedback inhibition of Gsh1, leading to increased GSH synthesis. In the case of severe GSH depletion, the hugely increased demand for GSH biosynthesis requires an increase in the levels of Gsh1, and therefore, GSH1 expression is increased via Yap1 activation.
The role of Met4 in regulating GSH homeostasis is less apparent. In a gsh1 mutant there is no induction of other Met4 target genes, indicating that there is not a general activation of Met4 and the sulfate assimilation pathway. Met4 is also known to regulate the response of GSH1 to cadmium stress, which additionally requires Yap1 (26). However, the cellular response to cadmium is different from the response to GSH depletion, since cadmium strongly induces the sulfate assimilation pathway (58,59). The transcriptional activation activity of Met4 is known to be regulated by ubiquitination, which affects its degradation and/or promoter recruitment (34). Specificity exists in this system, since for example, in rich media oligoubiquitinated Met4 is recruited to the promoters of genes re- FIG. 4. CDNB activates Yap1 and oxidizes thioredoxins. A, the expression of GSH1::lacZ, GSH1::lacZ minus the YRE, and YRE::lacZ reporter constructs was measured in exponentially grown wild-type treated with 0.08 mM CDNB for 2 h. B, to measure the redox state of thioredoxins, proteins were precipitated with trichloroacetic acid, and free thiols were modified by reaction with 4-acetamido-4Јmaleimidyldystilbene-2,2Ј-disulfonic acid. Samples were separated using 18% SDS-PAGE, and thioredoxins (TRX) detected by Western blot analysis. Fully oxidized (OX.) and fully reduced (RED.) proteins are indicated. Wild-type cells were grown into exponential phase in SD media and treated with 2 mM diamide or 2 mM CDNB. U, units. quired for S-adenosylmethionine biosynthesis but not to those of the sulfate assimilation pathway. The specificity of Met4 in GSH1 regulation could be explained by a similar mechanism in which Met4 is recruited to GSH1,whereas there is a selective repression of the other Met4-dependent genes. However, GSH1 can still be induced by GSH depletion in a met4 cbf1 mutant, indicating that Met4 plays an indirect role in the transcriptional activation of GSH1.
The GSH1 promoter contains two consensus Cbf1 binding sites, one of which (Ϫ367 to Ϫ362) is extremely close to the Yap1 binding site (Ϫ384 to Ϫ378) (15,26). The proximity of the Cbf1 and Yap1 binding sites indicates that promoter occupancy may be dependent on the presence of each factor. In support of this, GSH1 expression is strongly elevated in a cbf1 mutant but returns to basal levels in a cbf1 yap1 double mutant, indicating that Cbf1 may play a repressive role in the binding of Yap1 to GSH1. We propose that Met4 interacts with Cbf1 to overcome this inhibition. Thus, in a met4 mutant, Cbf1 repression cannot be overcome, and there is no induction of GSH1 expression after GSH depletion. When CBF1 is deleted, GSH1 can respond, regardless of the presence of Met4. A repressive role for Cbf1 in regulating transcription through promoter occupancy has been described previously in the response of QCR8, encoding a subunit of mitochondrial ubiquinol-cytochrome c oxidoreductase, to growth on a non-fermentable carbon source (60). The general DNA-binding protein Abf1 is required for QCR8 induction after an increased demand for mitochondrial biogenesis. Cbf1 and Abf1 have overlapping binding sites on the QCR8 promoter, and therefore, Cbf1 represses QCR8 transcription directly by preventing the binding of Abf1. An alternative to the promoter occupancy model is the possibility that Cbf1 regulates GSH1 expression through chromatin remodeling. Moreau et al. (61) recently demonstrated that Cbf1 is able to bind the Isw1 chromatin remodeling complex and target it to the promoters of PHO8 and PHO84. This leads to the displace-ment of the TBP (TATA-binding protein) and repression of the target gene. It is conceivable that Cbf1 plays a similar role in GSH1 expression and that an interaction with Met4 acts to overcome this. We are currently investigating the interactions between Cbf1 and Met4 at the level of the GSH1 promoter to understand how Cbf1 acts to repress its expression.
The requirement for Met4 allows the cell to couple Yap1mediated GSH1 expression to the availability of both GSH and methionine (Fig. 6). Thus, upon GSH depletion, Met4 interacts with Cbf1 to allow Yap1-mediated activation of GSH1 expression. However, in the presence of methionine, Met4 becomes ubiquitinated, and Cbf1 represses Yap1 activation of GSH1 in a manner similar to that seen in the met4 mutant. Methionine does not prevent Yap1 activation after exposure to CDNB, indicating that it specifically acts to repress GSH1 expression. Similarly, methionine did not repress expression in a cbf1 mutant, indicating repression occurs via Cbf1. This mechanism is distinct from the oxidative stress response of GSH1, which is independent of Met4 (62). It is not known how Yap1 overcomes Cbf1-mediated repression in the oxidative stress response, but the observation that the YRE is not required for GSH1 induction after H 2 O 2 exposure (17) indicates that there may be differing requirements for Yap1 binding in the two responses.
The ability of methionine to repress GSH1 expression after GSH depletion is unexpected. This is particularly true since methionine and other sulfur-containing compounds are unable to substitute for the essential function of GSH in cells (1,14). Upon exposure to a xenobiotic such as CDNB, which depletes GSH, there would clearly be an increased demand for GSH. One would expect that the addition of methionine would allow a ready supply of sulfur for GSH biosynthesis and, therefore, would stimulate rather than inhibit the synthesis of this sulfur storage compound. Presumably it must be unfavorable to synthesize more GSH in a sulfur-rich but GSH-depleted environment. This suggests that cells have a different strategy to cope FIG. 5. Addition of methionine represses GSH1 expression, but not Yap1 activation, in response to CDNB. The expression of the GSH1::lacZ and YRE::lacZ reporter constructs was determined in wild-type cells grown to exponential phase with or without the presence of 1 mM methionine. Cells were treated with 0.08 mM CDNB for 2 h to deplete the cellular GSH pool. U, units. Met4 couples Yap1-mediated GSH1 expression to the availability of GSH and methionine. Thus, when methionine concentrations are low (B) Yap1 will up-regulate GSH1 expression in response to GSH depletion. In contrast, high concentrations of methionine (A) will result in the inactivation of Met4, preventing the Yap1-regulated induction of GSH1 expression (other Yap1-controlled genes will be unaffected). This model is based on the idea that Cbf1 normally acts to repress GSH1 expression and GSH depletion signals Met4 to overcome this inhibition.
with GSH depletion in a sulfur-rich environment. One possibility is that the glutathione, which has been utilized in the GSH conjugation/detoxification pathway, is recovered from the extracellular media. Thus, during sulfur-rich conditions, GSH would be recovered from outside the cell rather than via de novo biosynthesis. To further understand this complex phenotype we have examined the gsh1 microarray data for any genes that may be regulated in a similar manner. Genes were identified that were up-regulated in a gsh1 mutant and contained both Yap1 and Cbf1 binding sites in their promoters. We identified four genes, three of which were also identified as upregulated during microarray analysis of a cbf1 mutant (microarray data are to be published elsewhere). We are currently investigating the mechanisms underlying the transcriptional regulation of these genes and their contribution to defense against GSH depletion. The co-regulation of GSH biosynthesis by Yap1 and Met4 represents a mechanism through which the cells can co-ordinate the assimilation of sulfate with the demands for defense against oxidants and xenobiotics. The identification of further targets for co-regulation will help us understand how different biological systems interact to protect the cells against environmental stresses.