Coupling of the transcriptional regulation of glutathione biosynthesis to the

Transcription

1 JBC Papers in Press. Published on September 26, 2003 as Manuscript M Coupling of the transcriptional regulation of glutathione biosynthesis to the availability of glutathione and methionine via the Met4 and Yap1 transcription factors Glen L. Wheeler, Eleanor W. Trotter, Ian W. Dawes 1 and Chris M. Grant* Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchester M60 1QD, U.K. 1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. *Corresponding Author: Dr Chris M. Grant, Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, U.K. Phone: (0161) Fax: (0161) chris.grant@umist.ac.uk Running Title: Regulation of GSH biosynthesis 1 Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

2 SUMMARY 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-glutamyl-cysteine 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 remain unaltered. Glutathione depletion appears to result in Yap1-activation via oxidation of thioredoxins, which normally act to downregulate 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-dependant transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression following 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 sulphur status of the cell through a specific Met4-dependant mechanism. 2

3 INTRODUCTION 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 prioritise the allocation of resources to support the increased demand for defensive mechanisms. Understanding the mechanisms which 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 biosynthesis of glutathione (GSH) in the yeast Saccharomyces cerevisiae. GSH is a tripeptide (γ-l-glutamyl-lcysteinylglycine) which plays an important role in protecting yeast cells against damage induced by oxidative stress (1-3). GSH counters the potentially damaging effect of reactive oxygen species (ROS) 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 sulphydryl groups can protect them from irreversible oxidative damage (6,7). There are also requirements for GSH in methylglyoxal detoxification, as a co-factor 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 γ- glutamylcysteine synthetase, encoded by GSH1 (9,10). This produces γ-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 Saccharomyces cerevisiae (1,13,14). The rate-limiting step in the biosynthetic 3

4 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 an oxidative stress, GSH1 expression is increased in a Yap1-dependant manner (15-17). Yap1 is a redox-sensitive bzip-transcription factor which 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 (NES) by an intramolecular disulphide bond (21,22). It has recently been shown that the glutathione peroxidase like-enzyme, Gpx3, is required for the H 2 O 2 dependant formation of the intramolecular disulphide 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) 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 which regulates the assimilation of extracellular sulphate into the sulphur 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 heteromeric complex involving the bzip-protein, Met28, and the bhlh 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 sulphate assimilation pathway have differing requirements for one or both of these complexes. The GSH1 4

5 promoter contains binding motifs for both Cbf1 and Met31/32 (26). The sulphate assimilation pathway is turned off upon exposure to methionine, which triggers the ubiquination of Met4 (32,33). The nature of this regulation is complex, as 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 present work, we have 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 following 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 defence against xenobiotics which deplete cellular GSH concentrations. 5

6 EXPERIMENTAL PROCEDURES Yeast strains, growth conditions and plasmids. The Saccharomyces cerevisiae strains used in this study are derivatives of CY4 (MATa ura3-52 leu2-3 leu2-112 trp1-1 his3-11 can1-100) (1). Strain CY197, which is deleted for GSH1, has been described previously (13), as have the yap1 mutant (36) and the met4 and gsh1 met4 mutants (35). Strain CY813, which is deleted for CBF1, was made by backcrossing CY4 with EUROSCARF strain Y16858 (MATα his3d1 leu2d0 lys2d0 ura3d0 cbf1::kanmx4). These strains were used to construct the gsh1 yap1, gsh1 cbf1, met4 cbf1, yap1 cbf1, gsh1 met4 cbf1 and gsh1 yap1 cbf1 mutants using standard yeast genetic methods. 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, 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. Where appropriate, media was further supplemented by a non-repressive concentration of methionine (0.05mM) to overcome the methionine auxotrophy of cbf1 and/or met4 mutants. Sensitivity to 1-chloro-2,4- dintrobenzene (CDNB) was determined by growing cells to stationary phase (48 h growth) and spotting onto agar plates. The plasmids used in this study containing the GSH1::lacZ (pydj73 and pydj76, (26) and YRE::lacZ (15) reporter constructs have been described previously. 6

7 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 thiol-reactive probe AMS (Molecular Probes) as described previously (38) Western blot analysis. Protein extracts were subjected to electrophoresis on 18% SDS-PAGE minigels and electroblotted onto a poly(vinylidene difluoride) membrane (Amersham Pharmacia Biotech). Blots were incubated in anti-trx antibody (1:1000 dilution) as described previously (39). Bound antibody was visualised by chemiluminescence (ECL, Amersham Pharmacia Biotech) following incubation of the blot in donkey anti-rabbit immunoglobulin-hrp conjugate (Santa Cruz, CA). β-galactosidase assays. For the determination of β-galactosidase activity, transformants were assayed essentially as described previously (40). Activity is expressed as nanomoles of o- nitrophenyl-β-d-galactopyranoside (ONPG) hydrolysed per minute per microgram of total protein (U). All β-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 standard errors. Northern Blot analysis. Yeast cells were grown to mid-exponential phase (A 600 = ) in minimal media, in the presence of 1mM methionine or 1mM GSH where appropriate. Total RNA was prepared by Trizol extraction according to the manufacturers specifications (Life Technologies). RNA (50µg) was electrophoresed on 1.2% agarose-formaldehyde gels, blotted 7

8 overnight onto a Nylon+ membrane and hybridised with 32 P-labelled DNA specific probes. DNA probes were synthesised by PCR using the following oligonucleotides: MET3 (MET3-1, 5 -GGGTCTCTCTCTGTCGTAACAGTTG-3', MET3-2, 5 - TTGAGATGGGAGCATTTTATGACGA-3'), MET16 (MET16-1, 5 CAAAGGTATCAACCCATAGCAACTC-3', MET16-2, 5 -CGTACAGCGCGA ATTCTCCGCCAGC-3') and MET25 (MET25-1, 5 -CAATTCTATTAC CCCCATCCATACA- 3', MET25-2, 5 -TAATTTTACAAC TCATTACGCACAC-3'). Probes were labelled with the Megaprime kit (Amersham). ACT1 was used as a control for RNA loading. Microarray hybridisations and data analysis. Yeast cells were grown in triplicate to midexponential 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. In order 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 OD 600 of Preparation of RNA, probes and hybridisation to whole yeast genome microarrays (YG-S98, Affymetrix) was performed as described on the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) website ( Data acquisition was performed using Affymetrix Microarray Suite v5.0 software and analysed 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 8

9 greater than two at the 90% confidence level, 2) the mean expression values are significantly different using an unpaired t-test (p<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 website ( and genome- respectively) (19,20). Genes exhibiting a log 2 expression ratio higher than 1.0 were compared to the data set obtained in the gsh1 mutant. The identification of prospective regulatory sequences within promoters was performed by RSAT (Regulatory Sequence Analysis Tools, 9

10 RESULTS The regulation of the sulphate assimilation pathway is not affected by cellular GSH concentrations. The regulation of metabolic pathways is essential for a cell s 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 dependant on the presence of the Met4 transcription factor (35). In order to understand the mechanisms regulating GSH1 expression, we were particularly interested in the transcriptional responses of other Met4-dependant genes in response to GSH depletion. We therefore analysed 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 ORF s significantly upregulated 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 (manuscript in preparation). The microarray analysis of the gsh1 mutant indicates that lower cellular GSH concentrations do not lead to a general induction of the sulphate assimilation pathway. There is no significant effect on the expression of genes involved in sulphur metabolism, other than a 3.3-fold increase in STR3 transcripts. The STR3 gene encodes cystathionine β-lyase, which catalyses the conversion of cysteine to cystathionine, and would thus prevent a toxic accumulation of cysteine in the gsh1 mutant (42). In order to confirm the effect of GSH depletion on the regulation of the sulphate assimilation pathway, we analysed the transcriptional response of MET3, MET16 and MET25 using northern blot analysis (Fig. 1A). These genes have previously been shown to be controlled 10

11 by the Met4 transcriptional activator protein (27). However, none of these genes were induced by GSH depletion in the gsh1 mutant, nor were they repressed by the exogenous addition of 1mM GSH. This is in strong contrast with the induction of GSH1 expression observed following GSH depletion (Fig. 1B). The addition of 1mM methionine severely repressed the expression of all three sulphate assimilation genes and also repressed the GSH1 induction observed in the gsh1 mutant. Thus, whilst 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 conditions. 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 which are regulated by the Yap1 transcriptional activator protein are upregulated in the gsh1 mutant (Table 1). 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 wholegenome expression data identified 16 genes in the gsh1 mutant, from a set of 35 genes in the trr1 mutant, which are strongly induced (greater than two-fold) in both mutants. The group of genes upregulated in a gsh1 mutant also contains 25 genes that have previously been shown to be upregulated (from a set of 118 genes) following the overexpression of plasmid-borne YAP1 in wild-type cells (20). Furthermore, analysis of the promoter regions of the genes upregulated in the gsh1 mutant indicates that a significant proportion (43 out of 151) contain a putative Yap1-binding site (TTAC/GTAA or TGACTAA). 11

12 In order 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 oxidised GSSG back to GSH and consequently the redox status of the GSH pool is much more oxidised (36). However, oxidation of the GSH pool does not result in Yap1 activation as no target genes were upregulated 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 Yap1-responsive elements (YRE) from the promoter of GSH1 fused upstream of the β-galactosidase reporter gene (15). The gsh1 mutant strain exhibited a five-fold increase in YRE::lacZ expression compared to 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 following 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 following GSH depletion. Loss of CBF1 removes the requirement for Met4 in GSH biosynthesis. In order 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 12

13 complex containing Met28 and either Cbf1 or Met31/32 (28,31). As a consequence, many genes of the sulphate 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 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 Yap1-dependant 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 synthesise GSH. We wished to confirm that a similar response occurs in wild-type cells which have been depleted of GSH and to ensure that the response is not specific to the gsh1 mutant. The GSH pool in wild- 13

14 type cells may be depleted following exposure to xenobiotics, such as CDNB, which are rapidly detoxified by GSH-conjugation (46). Thus, the response 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 to wild-type cells, indicating that Met4 is important in maintaining the GSH pool during normal aerobic growth (Table 2). 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 0.08mM CDNB for 2 hours 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 that caused by deletion of the GSH1 gene. Exposure to 0.08mM CDNB for 2 hours resulted in an increased expression of GSH1::lacZ (Fig. 3A). This response was dependent on both the Yap1 and Met4 transcription factors, as 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 14

15 regulation of GSH1 expression following 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 following 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 upregulate GSH biosynthesis is necessary for resistance to GSHdepleting agents Exposure to CDNB causes thioredoxin oxidation and Yap1-activation. Given that GSHdepletion 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 eight-fold upon CDNB treatment, indicating that Yap1 is strongly activated following 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 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 15

16 physiological GSH-depletion that occurs following exposure to CDNB could also affect the redox status of thioredoxins. Thioredoxin are predominately present in the reduced form in wildtype cells, but are shifted to a more oxidized form following treatment with CDNB (Fig. 4B). In contrast, treatment with the thiol oxidising 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 GSHdepletion, 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 coregulation of GSH1 by both Yap1 and Met4 in response to GSH depletion suggests that there is a need to balance GSH biosynthesis with the sulphur 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 tested whether Met4 acts to couple GSH biosynthesis to the cell s demand for sulphate assimilation. The addition of methionine strongly represses the transcription of genes required for sulphate assimilation through the inactivation of Met4. Similarly, methionine addition was found to abrogate the normal induction of GSH1 expression induced by CDNB treatment (Fig. 5A). 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. 5B). Thus, CDNB 16

17 causes Yap1 activation regardless of the presence of methionine, but only induces GSH biosynthesis when methionine is absent. 17

18 DISCUSSION Glutathione is an abundant and ubiquitous low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl 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 ROS (4,48-51). Not surprisingly therefore, GSH is an essential metabolite in eukaryotes. For example, mice which 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 haemolysis and defective brain function, cataract formation and oxidative damage to renal, hepatic and brain tissues (53-56). 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 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 coregulated by the Yap1 and Met4 transcription factors. Previous reports have indicated 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, since Yap1 is normally deactivated via thioredoxin-mediated reduction of an intramolecular disulphide bond (22). Oxidation of the GSH pool in the glr1 mutant did not cause any activation 18

19 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. Whilst 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 so 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 sulphate 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 to the response to GSH-depletion, since cadmium strongly induces the sulphate 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 oligo-ubiquitinated Met4 is recruited to the promoters of genes required for s-adenoysl methionine biosynthesis but not to those of the sulphate assimilation pathway. The specificity of Met4 in GSH1 regulation could be explained by a similar mechanism in which Met4 is recruited to GSH1 whilst there is a selective repression of the other Met4-dependant 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. 19

20 The GSH1 promoter contains two consensus Cbf1 binding sites, one of which (-367 to 362) is extremely close to the Yap1 binding site (-384 to 378) (15,26). The proximity of the Cbf1 and Yap1 binding sites indicates that promoter occupancy may be dependant 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 following 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 following an increased demand for mitochondrial biogenesis. Cbf1 and Abf1 have overlapping binding sites on the QCR8 promoter and so 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 remodelling. Moreau et al (61) recently demonstrated that Cbf1 is able to bind the Isw1 chromatin remodelling complex and target it to the promoters of PHO8 and PHO84. This leads to the displacement 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 in order to understand how Cbf1 acts to repress its expression. 20

21 The requirement for Met4 allows the cell to couple Yap1-mediated 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 ubiqutinated and Cbf1 represses Yap1 activation of GSH1 in a manner similar to that seen in the met4 mutant. Methionine does not prevent Yap1 activation following exposure to CDNB, indicating that it specifically acts to represses 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 following 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 following GSH depletion is unexpected. This is particularly true since methionine and other sulphur 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 sulphur for GSH biosynthesis and therefore would stimulate rather than inhibit the synthesis of this sulphur storage compound. Presumably it must be unfavourable to synthesise more GSH in a sulphur-rich but GSH-depleted environment. This suggests that cells have a different strategy to cope with GSH depletion in a sulphur-rich environment. One possibility is that the glutathione, which has been utilised in the GSH conjugation/detoxification pathway, is recovered from the extracellular media. Thus, during sulphur-rich conditions, GSH would be 21

22 recovered form outside the cell, rather than via de novo biosynthesis. In order to further understand this complex phenotype we have examined the gsh1 microarray data for any genes which may be regulated in a similar manner. Genes were identified which were upregulated 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 being upregulated during microarray analysis of a cbf1 mutant (microarray data to be published elsewhere). We are currently investigating the mechanisms underlying the transcriptional regulation of these genes and their contribution to defence against GSH depletion. The co-regulation of GSH biosynthesis by Yap1 and Met4 represents a mechanism through which the cell can co-ordinate the assimilation of sulphate with the demands for defence against oxidants and xenobiotics. The identification of further targets for co-regulation will help us understand how different biological systems interact to protect the cell against environmental stresses. Acknowledgements This work was supported by the Welcome Trust grant number (GLW) and BBSRC grant number 36/G13234 (EWT). We are grateful to Dr D. Jamieson and Dr S Moye-Rowley for donating plasmids and to Dr D. Thomas for the gift of the met4 mutant. We also thank the staff at the COGEME facility in Manchester University, UK, in particular Professor S. Oliver and Dr A. Hayes, for help with microarray analysis. 22

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28 Table 1. Comparison of gene expression profiles in a wild-type and gsh1 mutant identifies many genes which are regulated by Yap1. Gene name Accession Function Fold change 2µ YAP1 a trr1 b YRE c ARN1 YHL040C Siderophore transporter HSP26 YBR072W heat shock protein YOL153C YGR043C AAD6 YFL056C Putative aryl-alcohol dehydrogenase OYE3 YPL171C NAD(P)H dehydrogenase GTT2 YLL060C Glutathione transferase ECM4 YKR076W ExtraCellular Mutant DCS2 YOR173W GPM2 YDL021W phosphoglycerate mutase MRS4 YKR052C mitochondrial carrier protein YLL055W HXK1 YFR053C Hexokinase I RTN2 YDL204W member of the RTNLA subfamily YBR116C YBR116C AAD16 YFL057C putative aryl-alcohol dehydrogenase YKL071W NMA2 YGR010W nicotinamide adenylyltransferase YML131W YHR087W LAP4 YKL103C vacuolar aminopeptidase TMT1 YER175C Trans-aconitate Methyltransferase ALD4 YOR374W aldehyde dehydrogenase YOL048C YIL167W YMR090W YOL164W YLR108C ATR1 YML116W aminotriazole resistance ISU2 YOR226C Iron-sulphur cluster assembly FRE1 YLR214W Ferric reductase YLR046C YDL146W AAD4 YDL243C putative aryl-alcohol dehydrogenase VHT1 YGR065C vitamin H transporter GPH1 YPR160W Glycogen phosphorylase GRE2 YOL151W induced by osmotic stress COT1 YOR316C involved in cobalt accumulation DIT1 YDR403W first enzyme in dityrosine synthesis YDL124W YLR460C SDL1 YIL168W L-serine dehydratase

29 TSL1 YML100W trehalose-6-phosphate synthase YCR102C HAL1 YPR005C involved in halotolerance YNL134C GCY1 YOR120W Galactose-induced transcript MRL1 YPR079W Mannose 6-phosphate Receptor Like YGR154C YHR199C YDR533C AMS1 YGL156W Vacuolar alpha mannosidase YDL199C CVT19 YOL082W Required for protein-vacuolar targeting FLR1 YBR008C Major Facilitator Transporter PRX1 YBL064C mitochondrial thioredoxin peroxidase Genes included in this list fulfil one or more of the following criteria; a Elevated expression in cells containing YAP1 on a 2µ plasmid (20). b Elevated expression in a trr1mutant (19) c Contain a putative Yap1 binding site (YRE) in their promoter, either the TTAG/CTAA motif identified in TRX2 (21) or the TTAGTCA motif from GSH1 (15). 29

30 Table 2. The pool of cellular glutathione is depleted following CDNB exposure. control a CDNB a Strain GSH (nmol A -1 ml -1 ) GSSG (nmol A -1 ml -1 ) GSSP (nmol A -1 ml -1 ) GSH (nmol A -1 ml -1 ) GSSG (nmol A -1 ml -1 ) GSSP (nmol A -1 ml -1 ) Wild type 9.5 ± ± ± ± ± ± 0.03 met4 5.0 ± ± ± ± ± ± 0.01 cbf ± ± ± ± ± ± 0.10 met4 cbf ± ± ± ± ± ± 0.07 a Strains were either untreated or treated with 0.08 mm CDNB for 2 hours. Concentrations are shown for reduced (GSH), oxidized (GSSG) and protein-bound (GSSP) glutathione 30 Downloaded from by guest on November 12, 2018

31 FIGURE LEGENDS Fig. 1. The sulphate 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 1mM methionine (Met), 1mM 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 and gsh1 mutant strains. β-galactosidase activity was determined for exponential phase cultures grown in minimal media with no addition (control), 1mM GSH or 1mM methionine. 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 and gsh1 mutant strains which had been deleted for MET4 or YAP1. The expression of a GSH1::lacZ reporter construct lacking the Yap1-responsive element (YRE) was determined in wild-type and gsh1 strains. B. GSH1 expression was measured in wild-type and gsh1 mutant strains which had been deleted for CBF1, MET4 and CBF1 or YAP1 and CBF1. GSH1 expression was also determined in wild-type and gsh1 strains which had been deleted for CBF1 grown in the presence of 1mM methionine. 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, yap1, met4, cbf1 and met4 cbf1 strains following treatment with 0.08 mm CDNB for 2 hours. B. Sensitivity to CDNB was determined by spotting strains onto SD plates containing 31

32 0.02 mm or 0.03 mm CDNB. Cultures of wild-type, met4, cbf1 and met4 cbf1 strains were grown into stationary phase and adjusted to A , 0.1 and 0.01 before spotting onto plates. Growth was monitored after 3 days. Fig. 4. CDNB activates Yap1 and oxidises 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 hours. B. To measure the redox state of thioredoxins, proteins were precipitated with TCA and free thiols modified by reaction with AMS. Samples were separated using 18% SDS-PAGE and thioredoxins 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. Figure 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 1mM methionine. Cells were treated with 0.08 mm CDNB for 2 hours to deplete the cellular GSH pool. Fig. 6. Model for the co-regulation of glutathione biosynthesis by Yap1 and Met4 following glutathione depletion 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 32

33 (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. 33

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40 Coupling of the transcriptional regulation of glutathione biosynthesis to the availability of glutathione and methionine via the Met4 and Yap1 transcription factors Glen L. Wheeler, Eleanor W. Trotter, Ian W. Dawes and Chris M. Grant J. Biol. Chem. published online September 26, 2003 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts