A Genetic Investigation of the Essential Role of Glutathione

MUTATIONS IN THE PROLINE BIOSYNTHESIS PATHWAY ARE THE ONLY SUPPRESSORS OF GLUTATHIONE AUXOTROPHY IN YEAST*

Daniel SpectorDagger §, Jean LabarreDagger , and Michel B. ToledanoDagger §||**

From the Dagger  Service de Biochimie et Génétique Moléculaire, Bât. 142, Commissariat à l'Energie Atomique, F-91191 Gif-sur-Yvette Cedex, France, || Department of Pharmacology and Toxicology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855, § Graduate Program in Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences, Piscataway, New Jersey 08854

Received for publication, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of Delta gsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of Delta gsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that gamma -glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a Delta gsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glutathione (GSH) is a broadly conserved tripeptide with a highly reactive thiol and a very low redox potential of about -240 to -250 mV. Its elevated concentration, up to 10 mM, and the fact that its reduced state is efficiently maintained by NADPH-dependent glutathione reductase (reviewed in Refs. 1-3) confers to this small molecule the properties of a cellular redox buffer. As a redox buffer, GSH is thought to be a major determinant in maintaining a reducing cellular thiol-disulfide balance. GSH is also important as an electron donor for several enzymes that have a reducing step in their catalytic cycle, such as ribonucleotide reductase (4). In these situations, electrons from GSH are generally transduced to their substrates through glutaredoxins. GSH may protect protein sulfhydryls from irreversible oxidation by glutathionylation (5, 6). It also participates in the detoxification of peroxides, either directly or indirectly as a cofactor of glutathione peroxidase (1, 7, 8) and of several chemicals such as cadmium (9, 10).

In Saccharomyces cerevisiae and in probably all other GSH-containing organisms, GSH is synthesized in two steps beginning with the action of gamma -glutamyl cysteine synthetase (GSH1) (11, 12), which catalyzes the condensation of glutamic acid to cysteine, in the rate-limiting step of this biosynthetic pathway. The product of this reaction is gamma -glutamylcysteine, which is combined with glycine through the action of glutathione synthase (GSH2) (13) to form GSH. Null mutations in GSH1 result in GSH auxotrophy (14-16), demonstrating that GSH is essential in yeast. In mammals, GSH is also essential, as demonstrated by the embryonic lethality resulting from disruption of gamma -glutamyl cysteine synthetase and by the inability of blastocysts isolated from such knock-out strains to grow, except in the presence of GSH or the GSH substitute N-acetyl cysteine (17). The reason for the essential requirement of GSH in eukaryotes is not known. In contrast, GSH is not essential in prokaryotes since strains of Escherichia coli lacking gshA, the homologue of GSH1, can grow in minimal media without GSH supplementation (18-20).

We wished to understand the basis for the differential requirement of GSH between eukaryotes and prokaryotes by attempting to identify which of the functions of GSH might determine its essential role in yeast. To this end, we investigated whether mutations capable of suppressing the Delta gsh1 auxotrophy exist in yeast. We found that mutations of the proline pathway are the only suppressors of the lethal phenotype of the Delta gsh1 strain under both aerobic and anaerobic conditions. However, this suppression involves the biosynthesis of a small amount of GSH through a modified proline biosynthesis pathway, demonstrating that there is no single gene mutation able to bypass the yeast GSH requirement. Our data indicate that this requirement is not related to ribonucleotide reduction or to preventing toxic accumulation of non-native protein disulfide, leaving open this question. We used this strain as a model to study the contribution of GSH to oxidative stress tolerance.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Growth Conditions-- Y252 (MATa ura3-52 lys2-801amber ade2-101ochre trp1-Delta 1 leu2-Delta 1) and Y253 (MATalpha ura3-52 lys2-801amber ade2-101ochre his3-Delta 1 leu2-Delta 1) are derived from YPH98 (21) and were used throughout this study. The Delta yap1, Delta ahp1, and Delta tsa1 strains were described (22). Cells were grown in YPD1 (1% yeast extract, 2% peptone, and 2% glucose) or in minimal media (SD) (0.67% yeast nitrogen base w/o amino acids, 2% glucose) with casamino acid, tryptophan, uracil, adenine, and GSH supplements as appropriate. For anaerobic growth, plates containing SD supplemented with ergosterol (30 mg/liter) and Tween 80 (0.2%), were incubated in an anaerobic chamber in which the oxygen had been removed with a Merck Anaerocult A cassette, as confirmed by an indicator strip color change. With the exception of Delta gsh1PRO2-2/Delta glr1, the strains used in these assays were all respiration-competent, as shown by their ability to grow on YPG media that contains glycerol as the sole carbon source (not shown).

Ethylmethanesulfonate Mutagenesis and Mutant Selection-- Approximately 2 × 108 cells from an overnight culture were exposed to 3% ethylmethanesulfonate (Sigma) for 25-30 min at 30 °C, followed by a 4 h recovery in SD. Ethylmethanesulfonate-treated cells were spread on SD plates lacking GSH at a density of 5 × 106 cell/plate to select for mutants suppressing the GSH auxotrophy.

Genomic Library Construction-- Genomic DNA was isolated from the ySOG1-2 strain with the Qiagen blood and cell culture DNA kit and partially digested with Sau3AI. 8-15-kilobase-size fragments of the digested DNA were purified with the Qiagen QiaexII Gel extraction kit after separation on an agarose gel and ligated into the BamHI site of centromeric vector Ycp50. 40% of the E. coli transformants contained an insert, resulting in a library consisting of ~10,000 independent clones.

Gene Disruptions and Plasmid Constructions-- The PRO2 allele isolated from the library was amplified by polymerase chain reaction using the following primers, (5'-CCCGGATCCATTTCTTTTGATGGGCGGC-3', 5'-CCCGGATCCTCGAGTCAGCTCAAATCGCAT-3') and ligated into the BamHI site of Ycp50 to create pPRO2-1. The wild type PRO2 expression vector, PRO2, and PRO1 disruption cassettes were gifts from Dr. Brandriss (23). The Delta gsh1 null mutant was created by replacing 180 base pairs upstream and 500 base pairs downstream of the GSH1 ATG with the LEU2 gene. Disruption was confirmed by Southern blot. Oligonucleotide-mediated gene replacement was used to replace the entire coding sequence of GLR1 with the Kluyveromyces lactis URA3 gene and of GPX3 with the TRP1 gene.

Sensitivity Assays-- For the patch assay, cells were grown overnight to saturation in YPD, diluted in distilled H2O, and patched (2000 cells/10 µl) on plates containing various concentrations of H2O2 or t-butyl hydroperoxide. Plates were incubated for about 72 h at 30 °C. For the challenge assay, exponentially growing cells (A600 = 0.3-0.5) in SD were exposed to various concentrations of H2O2 for 1 h and then plated on YPD to determine the number of colony-forming units.

Measurement of GSH by a Microbiological Assay-- Cells (50 ml) grown in SD to an A600 = 2 were collected by centrifugation, resuspended in distilled H2O (2 ml), and lysed by alternately placing in boiling water 10 min and freezing in dry ice/ethanol for three cycles. Samples were centrifuged and the supernatant, which contained the total free cellular GSH (which included the cytosolic, endoplasmic reticulum and vacuolar pools) was analyzed for its ability to complement the growth of the Delta gsh1 strain previously depleted of its GSH content by overnight growth in GSH free media.

Analysis of GSH by Thin Layer Chromatography-- For the measurement of GSH in Delta gsh1PRO2-1, cells were grown in SD to an A600 = 0.4 and pulse-labeled with [35S]Met (150 µCi) for 1 h or for 3 h with a second [35S]Met pulse at t = 1 h 30 min, centrifuged, resuspended in 30 µl of distilled H2O, and heated for 10 min at 99 °C. Cell debris was centrifuged, and the supernatant, which contained the metabolic pool, was mixed with an equal volume of performic acid (90% formic acid, 10% H2O2) to resolve GSH into a single oxidized spot. 0.2 µl of each sample was dropped on a thin layer chromatography plate and allowed to migrate twice 6-7 h in the presence of 60% butanol, 10% acetic acid. The dried plate was exposed to autoradiography. Measurement of the GSH pool and GSH biosynthesis rate was performed similarly except that cells were grown in synthetic medium (SM) with glucose (5 g/liter), tryptophan, uracil, lysine, leucine, histidine, and adenine instead of casamino acids (A600 = 0.4) and labeled with [14C]glucose (150 µCi/ml) or [14C]glutamate for the indicated time. For the measurement of GSH export, 1 mCi of [35S]Met was added to cells and grown in SM (1.2 ml, A600 = 0.4) for 60 min. Cells were washed and resuspended in SM/distilled H2O (1:9, vol/vol) supplemented with 2% glucose and amino acids, and the supernatant was analyzed for GSH content at the indicated times after evaporation in a SPEEDVAC to <FR><NU>1</NU><DE>30</DE></FR> original volume and then treatment with an equal volume of performic acid.

DNA Content Determination by Fluorometry-- To examine growth-arrested cells, Delta gsh1 was depleted of GSH by overnight growth in GSH-free SD medium. Cells were fixed with 70% ethanol, washed in phosphate-buffered saline pH 7.4, treated with 1 mg/ml RNase A for 1 h, stained with propidium iodide, and analyzed by flow cytometry on a FACScalibur. Exponentially growing wild type and Delta gsh1 supplemented with GSH were used as controls.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phenotypes of the gsh1 Null Strain-- The Delta gsh1 strain is a glutathione auxotroph on minimal (SD) but not on rich media (YPD) (14-16), which contains GSH. After GSH withdrawal, the auxotrophic strain underwent growth arrest after about seven generations, forming large unbudded cells followed by a loss of viability after 3-4 days (not shown). GSH, whether in the reduced (14, 15) or oxidized form, supported growth of the auxotrophic strain at a concentration as low as 0.5 µM, as did the reduced thiol dithiothreitol (15) at the mM range concentration (Fig. 1A). However, anaerobic conditions did not rescue the GSH auxotrophy but only delayed growth arrest by several generations (not shown). Similarly, cysteine only delayed growth arrest of a Delta gsh1 strain that had not been depleted of GSH and had no effect upon cells that had been previously depleted of GSH by overnight growth in SD media lacking GSH (Fig. 1A). GS-CH3, a GSH derivative with a methyl group replacing the sulfhydryl, did not support growth either (not shown). Microscopic analysis showing a predominance of unbudded cells after GSH withdrawal (not shown) and the analysis of the cellular DNA content by fluorocytometry (Fig. 1B) suggested that cells arrested predominantly at the G1 phase of the cell cycle. This observation and the inability of anaerobic conditions to rescue the GSH auxotrophy strongly suggest that the essential requirement of GSH is not related to ribonucleotide reduction or to preventing a toxic accumulation of non-native protein disulfides (see "Discussion").



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Fig. 1.   Phenotypic consequence of GSH depletion. A, the growth of Delta gsh1 is rescued by GSH or dithiothreitol (DTT) but not by cysteine or methionine. The Delta gsh1 strain not depleted (Delta gsh1) or depleted of GSH by overnight growth in SD media devoid of GSH (Delta gsh1 depleted) was patched on SD medium containing GSH, dithiothreitol, cysteine, or methionine at the indicated concentrations. B, Delta gsh1 arrests in G1 after GSH withdrawal. The DNA content of exponentially growing wild type, Delta gsh1 grown in medium containing GSH (Delta gsh1), and Delta gsh1 growth-arrested due to GSH depletion (Delta gsh1 depleted) was measured by flow cytometry after propidium iodide staining.

Specific Mutations in PRO2 Can Suppress GSH Auxotrophy-- Given the apparently essential nature of GSH, we wished to determine whether it was possible to isolate mutations that suppressed the GSH requirement of the Delta gsh1 strain. Using ethylmethanesulfonate mutagenesis, about 40 mutants able to grow on SD without GSH were isolated in each mating type of the Delta gsh1 strain and labeled SOG (suppressor of GSH auxotrophy). Complementation analysis showed that nine of the mutants were dominant and that all of the recessive mutations were in the same locus. Two dominant (ySOG1-1, ySOG1-2) and 1 recessive mutant (ysog1-3) were further analyzed. After failing to clone the recessive mutant by library complementation, a genomic library was constructed from the dominant mutant ySOG1-1. Several plasmids able to suppress the GSH auxotrophy of the Delta gsh1 strain were isolated from the ySOG1-1-derived genomic library, each containing a related genomic insert. Sequencing one of these inserts showed that it contained an allele of the PRO2 gene, which encodes gamma -glutamyl phosphate reductase. The suppression phenotype was further mapped to the PRO2 locus by the ability of the PRO2 allele polymerase chain reaction amplified from the isolated genomic library clone and referred to as PRO2-1 to confer the suppression phenotype to the Delta gsh1 strain and by the loss of the ySOG1-1-suppressed phenotype after null disruption of PRO2. The sog recessive suppressor mutations were also mapped to the PRO2 locus by the loss of the suppression phenotype of ysog1-3 upon either expression of wild type PRO2 or deletion of the corresponding locus. Sequencing of PRO2-1 and of the PRO2 alleles of ySOG1-2 and ysog1-3 showed that each contained a single base substitution corresponding to S124P, G148S, and A226V, respectively. The suppressor mutation was established in a fresh background by integrating the PRO2-1 allele at the PRO2 locus in Delta gsh1, resulting in strain Delta gsh1PRO2-1.

Suppression by Alleles of PRO2 Requires gamma -Glutamyl Phosphate-- PRO2 encodes gamma -glutamyl phosphate reductase, which catalyzes the second step of the proline biosynthetic pathway. The first step of this pathway is catalyzed by gamma -glutamyl kinase (PRO1), which converts glutamate to gamma -glutamyl phosphate (23). The PRO2 gene product then converts gamma -glutamyl phosphate to L-glutamate-gamma -semialdehyde, the precursor of L-Delta 1-pyrroline-5 carboxylic acid, which is converted to proline by the PRO3 gene product. To further examine the role of this pathway in the suppression phenotype, we disrupted PRO1 in both Delta gsh1PRO2-2 and ysog1-3. This restored GSH auxotrophy in both strains, demonstrating that the mechanism of suppression requires gamma -glutamyl phosphate as an obligate substrate of the suppressor alleles of PRO2. Note that none of the PRO2 suppressor alleles analyzed had the proline auxotrophy of the null mutations of this gene (23), demonstrating that their proline biosynthetic pathway remained functional (not shown).

We wished to determine whether any suppressors of GSH auxotrophy existed independently of the proline pathway. To this end, we performed a mutagenesis in the Delta gsh1Delta pro1PRO2-1 background. This mutagenesis failed to isolate any new suppressor under either aerobic or anaerobic conditions, leading to the conclusion that this pathway is probably the only mechanism that can suppress the GSH auxotrophy of the Delta gsh1 strain.

Suppression by Alleles of PRO2 Involves the Synthesis of Trace Amounts of GSH-- Since the suppression of the GSH auxotrophy by alleles of PRO2 could involve the synthesis of GSH, the Delta gsh1PRO2-1 strain was analyzed for its GSH content. This strain grew slightly slower than the wild type strain in SD, but the slow growth rate was corrected by the addition of GSH (Fig. 2A). Unlike the wild type strain, which can support the growth of the Delta gsh1 strain when grown in its vicinity, presumably by releasing GSH into the medium, Delta gsh1PRO2-1 failed to cross-feed Delta gsh1 (Fig. 2B). These data indicate that if Delta gsh1PRO2-1 contains GSH, it is present in very limiting quantity. We more directly analyzed the presence of GSH by thin layer chromatography (TLC) of the yeast metabolic pool after [35S]methionine metabolic labeling (Fig. 2C). GSH was visible as a discrete spot in wild type but not in Delta gsh1 cell extracts. In Delta gsh1PRO2-1 cells, glutathione was not visible after a 1-h labeling but could be detected after a 3-h labeling. To further quantify the presence of GSH in Delta gsh1PRO2-1, we used an assay based upon the ability of cell extracts to support the growth of Delta gsh1 (Fig. 2D). Delta gsh1PRO2-1 cell extracts could support the growth of Delta gsh1 with an efficiency similar to that of wild type extracts diluted 150-fold. This confirmed the presence of GSH in Delta gsh1PRO2-1 at levels about 0.5-1% that of wild type levels. The growth support provided by this extract could not be due to cysteine, because cysteine is not a GSH substitute (see Fig. 1A). This demonstrates that the mechanism of suppression by alleles of PRO2 involves the synthesis of small amounts of GSH.



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Fig. 2.   Delta gsh1PRO2-1 contains low but measurable levels of GSH. A, growth curves of wild type (WT, black diamond) and Delta gsh1PRO2-1 (gray square) grown in SD and Delta gsh1PRO2-1 grown in SD containing GSH (gray triangle). B, the wild type strain but not Delta gsh1PRO2-1 can support growth of the Delta gsh1 GSH auxotrophic strain by releasing GSH in the medium. Delta gsh1 was spotted (2000 cells/10 µl) on a SD plate adjacent to the wild type (wt) or to the Delta gsh1PRO2-1 strain. Growth was recorded after 3 days of incubation at 30 °C. C, autoradiogram of a TLC plate that separated the metabolic pool of the wild type (wt, lanes 1 and 2), Delta gsh1 (lanes 3 and 4), and Delta gsh1/PRO2-1 (lanes 5 and 6) strains labeled with [35S]methionine for 1 (lanes 1, 3, and 5) or 3 h (lanes 2, 4, and 6). Extracts were oxidized with performic acid to resolve GSH into a single oxidized spot (see the brackets). D, measurement of GSH by a microbiological assay. Cells from a culture of Delta gsh1 or Delta gsh1PRO2-1 were spotted (2000 cells/10 µl) as indicated on the figure onto SD plates containing 3, 5, 20, 100, 500, or 1000 µl of crude extracts prepared from the wild type (wt) or the Delta gsh1PRO2-1 strains. Growth was recorded after 3 days of incubation at 30 °C.

GLR1 Is Not Essential for the Suppression Phenotype-- Since the Delta gsh1PRO2-1 strain contains very low levels of GSH, we considered that its ability to reduce GSSG might become essential for cell viability. Therefore, we disrupted the GLR1 gene in wild type, Delta gsh1, and Delta gsh1PRO2-1. Despite the absence of glutathione reductase, the Delta gsh1PRO2-1Delta glr1 strain retained the ability to grow on minimal media without GSH supplement. Interestingly, we also observed that the growth of Delta gsh1Delta glr1 could be supported by GSSG (data not shown), suggesting that either an alternate mechanism for GSSG reduction exists in yeast or, less likely, that GSSG can somehow perform the essential role of GSH in yeast.

High Levels of GSH Are Essential for Acute but Not Chronic Oxidative Stress-- Previous examinations of the contribution of GSH to the tolerance to oxidative stress could have been limited by the GSH requirement of the Delta gsh1 strain. The Delta gsh1PRO2-1 strain seemed in this regard an appropriate model. On YPD plates (Fig. 3, A and B), the Delta gsh1PRO2-1 strain had a wild type H2O2 (A) tolerance but was slightly sensitive to t-butyl hydroperoxide (B, t-BOOH). On SD plates (C and D), the H2O2 (C) but not the t-BOOH (D) tolerance was slightly decreased (Fig. 3, C and D). Delta glr1 is hypersensitive to peroxide (24), and its deletion in Delta gsh1PRO2-1 (Delta gsh1PRO2-1Delta glr1) further decreased the peroxide-sensitive phenotype of both mutations (A-D). To further evaluate the contribution of the GSH pathway to the defense against peroxide, we compared the H2O2 tolerance of Delta gsh1PRO2-1 to that of strains lacking either the oxidative stress response regulator Yap1 (25, 26), the thiol peroxidases Tsa1 (27), Ahp1 (22, 28), or the GSH peroxidase Gpx3 (29). In all conditions tested, Delta gsh1/PRO2-1 was the least sensitive to peroxide among these strains, clearly demonstrating that normal levels of GSH are not critical in this response as measured by the plate assay.



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Fig. 3.   GSH is not essential to peroxide tolerance on solid medium. A-D, tolerance to peroxide of wild type (wt), Delta gsh1PRO2-1, Delta glr1, Delta gsh1PRO2-1Delta glr1, Delta yap1, Delta tsa1, Delta hyr1, and Delta ahp1 was evaluated by the patch assay on YPD (B and D) and on SD plates (C and E) containing the indicated amounts of H2O2 (B and C) or t-butyl hydroperoxide (t-BOOH) (D and E).

We also examined the tolerance to H2O2 by a challenge assay in liquid medium. In contrast to the plate assay, Delta gsh1PRO2-1 was considerably more sensitive than wild type (Fig. 4). Since the challenge assay measures the cell tolerance to an acute exposure to high levels of H2O2, whereas the plate assay examines the growth adaptation to a moderate concentration of this oxidant, these results might indicate that GSH is more important for acute stress rather than for the adaptation to H2O2.



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Fig. 4.   GSH is important to peroxide tolerance in liquid medium. The tolerance of wild type (wt, black diamond), Delta gsh1PRO2-1 (black square), Delta glr1 (gray triangle), and Delta gsh1PRO2-1Delta glr1 (gray circle) was evaluated by the challenge assay in SD medium. Exponentially growing cells (A600 = 0.3-0.5) in SD were exposed to various concentrations of H2O2 for 1 h and then plated on YPD to determine the number of colony-forming units.

Decreased GSH Synthesis Rate during Adaptation to H2O2-- As another means of evaluating the importance of GSH during the H2O2-adaptive response, we measured its biosynthesis rate by pulse-labeling cells for 5 min after exposure to 0.6 mM H2O2 (Fig. 5A). Surprisingly, compared with untreated cells, the amount of labeled GSH was significantly lower both 20 and 50 min after H2O2 exposure, suggesting either a decrease in the GSH biosynthesis rate or a release of GSH in the extracellular milieu. However, the GSH pool, estimated by labeling cells for 2 h before exposure to H2O2, was similar in untreated cells and in cells that had been exposed for 20 min to H2O2 (Fig. 5B). As another control, exposure to 0.6 mM H2O2 during either 20 or 50 min did not increase the GSH levels in the extracellular milieu (Fig. 5C), demonstrating that the decrease of 5-min pulse-labeled GSH after exposure to low H2O2 concentrations is most likely due to a decreased synthesis rate. Since GSH1 is induced by H2O2 (30, 31), the decreased synthesis rate of GSH after exposure to H2O2 could be related to a decreased availability of cysteine, as this amino acid can be limiting for GSH synthesis (32). The addition of cysteine to the medium indeed significantly increased to the same extent the GSH synthesis rate of untreated and H2O2-treated cells (Fig. 5D), suggesting that this amino acid is limiting for GSH biosynthesis, especially under oxidative stress conditions.



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Fig. 5.   The GSH biosynthesis rate decreases after H2O2 treatment. A, measurement of the GSH biosynthesis rate. Exponentially growing wild type cells in SM (A600 = 0.4) not exposed (lanes 1 and 3) or exposed to H2O2 (0.6 mM) (lanes 2 and 4) at t = 0 were pulse-labeled with [14C]glucose at t = 5 min and harvested at t = 20 min (lanes 1 and 2) or t = 50 min (lanes 3 and 4). Cells were lysed as indicated in the method, and the extract was separated on a TLC plate. B, measure of the GSH pool as described in A except that cells had been labeled with [14C]glucose at t = 0 and not exposed (lane 1) or exposed to H2O2 (lane 2) at t = 2 h and harvested at 2 h 15 min. C, measurement of GSH export after H2O2 treatment. Cells grown in SM (A600 = 0.4) and labeled during 1 h with [35S]Met were subsequently not exposed (lanes 1 and 3) or exposed (t = 0) (lanes 2 and 4) to H2O2 (0.6 mM). The supernatant of each sample was analyzed for its GSH content at t = 20 min (lanes 1 and 2) and t = 50 min (lanes 3 and 4). Purified labeled GSH was used as a control of GSH migration (lane 5). D, cysteine is limiting for GSH biosynthesis in the presence of H2O2. Cells were grown in SM in the absence (lanes 1 and 2) or in the presence of cysteine and glycine (400 µM) (lanes 3 and 4) and labeled with [14C]glutamate. 3 min after labeling, cells were not exposed (lane 1 and 3) or exposed (lanes 2 and 4) to H2O2, and the GSH content was analyzed at 20 min.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The essential nature of GSH in yeast (14-16) and in mammals (17) but not in prokaryotes provides an interesting puzzle. To address this difference, we designed a genetic selection for mutations that could bypass the requirement for GSH in yeast. We found that mutations in the proline biosynthetic pathway could suppress this requirement but actually did so by restoring the synthesis of GSH. These mutations were unique, since a second screen after ablation of the proline pathway did not yield any other suppressor under either aerobic or anaerobic conditions. Therefore, no single gene mutation can suppress the GSH growth requirement, suggesting that more than one mutation might be needed or, alternatively, that GSH is absolutely essential in yeast, and there is no genetic condition that can suppress this requirement.

Abduction of the Proline Pathway for GSH Biosynthesis-- The suppression by alleles of PRO2 involves the biosynthesis of small quantities of GSH. Although not very efficient, the modified proline pathway provides about 0.5-1% wild type GSH content, which is therefore a quantity compatible with the viability of the strain. What could then be the GSH biosynthetic mechanism by the modified proline pathway? Deletion of either PRO1 or PRO2 abolishes the suppression phenotype, demonstrating that formation of gamma -glutamyl phosphate is required for the suppression as an obligate substrate for the modified PRO2 allele. It is thus probable that the PRO2 suppressor alleles allow condensation of cysteine to the enzyme-bound gamma -glutamyl phosphate to produce gamma -glutamylcysteine. However, it is not clear how the addition of cysteine to gamma -glutamyl phosphate occurs, whether spontaneous, catalyzed by mutant Pro2p, or perhaps by a transpeptidase. The suppression of the GSH auxotrophy by alleles of PRO2 illustrates how a biosynthetic pathway can be genetically diverted from its natural role to fulfill another function.

The Essential Role of GSH-- In bacteria, only the simultaneous inactivation of both the GSH or the thioredoxin pathways results in aerobic inviability due to defects in protein thiol maintenance and/or ribonucleotide reduction (33, 34). However, anaerobic conditions restore growth because of the presence of an E. coli alternative ribonucleotide reductase that is independent of disulfide reduction (35) and also by the likely decrease in non-native disulfide bond formation. In contrast, GSH is essential in yeast (Refs. 14-16 and this work). This essential function is not related to ribonucleotide reduction since Delta gsh1 arrests in G1 with predominantly unbudded cells after GSH withdrawal, whereas a strain with a defect in ribonucleotide reduction and, therefore, DNA synthesis would be expected to arrest with large buds in S phase (36). In addition, the failure of anaerobic conditions to rescue the viability of Delta gsh1 indicates that its lethal phenotype is probably not related to oxidative stress or to toxic accumulation of non-native protein disulfides. Furthermore, in the latter case, it is expected that thioredoxin would compensate for the lack of GSH. The GSH auxotrophy is also not due to a defect in sulfate assimilation since methionine and cysteine cannot rescue the growth defect of Delta gsh1. What could then be the essential function of GSH in yeast? This essential function is clearly dependent on the redox properties of GSH, since it can be replaced by a thiol such as dithiothreitol (15) but not by a GSH analog with a methyl group instead of a thiol. Since GSH is the reductant for glutaredoxin and glutaredoxin is also essential (37), their essential function(s) might be the same. One or more essential enzymes requiring the reduction of a disulfide bond to complete their catalytic cycle might be exclusively dependent on the GSH reductive pathway in yeast. A search for the cellular targets of glutaredoxin might help us understand the essential requirement for GSH in yeast.

The Role of GSH in the Protection against Oxidative Stress-- Yeast cells depleted of GSH were shown to be hypersensitive to peroxides and to superoxide-generating drugs (13, 15, 16, 32). This together with the inducibility of GSH1 expression by oxidants suggested that GSH provides an important defense mechanism against oxidative stress (3, 8). We reevaluated this question with Delta gsh1PRO2-1 and observed that it was indeed sensitive to H2O2 by the 1-h challenge assay in liquid medium. However, when measured by the plate assay, Delta gsh1PRO2-2 had a near wild type H2O2 tolerance on YPD (Fig. 3A) and was just slightly sensitive to this oxidant on SD (Fig. 3C). Still, Delta gsh1PRO2-1 was moderately sensitive to t-butyl hydroperoxide but not as sensitive as strains lacking the Yap1 regulator or the peroxidases encoded by GPX3 or AHP1. The difference regarding the peroxide tolerance of strains with low GSH intracellular levels between the reported literature and this study might be accounted for by the fact that the Delta gsh1 strain used in those studies was respiratory-deficient, which by itself lowers the tolerance to oxidative stress (38), whereas Delta gsh1PRO2-1 was respiration-proficient. We conclude that high levels of GSH are important for surviving acute peroxide stress, as measured by the challenge assay, but not for growth adaptation under moderate peroxide concentrations, as measured by the patch assay. An inverse situation was observed with a strain lacking the regulator of the H2O2-adaptive response Yap1, which was hypersensitive to H2O2 in the patch assay but had a wild type resistance to this oxidant in the challenge assay (16). In addition, we observed that, although GSH1 transcription is induced by H2O2 (30, 31), the GSH synthesis rate decreases upon exposure to H2O2 when cysteine is limiting, suggesting that an increase in GSH might not be essential during adaptation to H2O2. The importance of GSH during an acute exposure to H2O2 might be related to its redox buffering properties. However, during adaptation to H2O2, the GSH levels might not be limiting for its function as a cofactor for glutathione peroxidase, allowing nearly normal detoxification of H2O2 provided that it is kept reduced by glutathione reductase. Indeed, deletion of GLR1 in Delta gsh1PRO2-2 resulted in a drop in H2O2 tolerance which demonstrated that, despite very low levels, GSH could still function in the scavenging of H2O2 in this strain.


    ACKNOWLEDGEMENTS

We thank Dr. Brandriss for kind gifts of plasmids, André Sentenac for continuous support, and Carl Mann and Christophe Leroy for helpful suggestions and help with flow cytometry. We thank members of the Toledano group for enthusiastic discussions and advice.


    FOOTNOTES

* This work was supported by a grant from Associatíon de Recherche Centre de Cancer (9575) and from the Ministère de l'Éducation (to M. B. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a National Institutes of Health predoctoral fellowship and of a Bourse de Formatíon Doctorale pour Érrangers/ Commissariat à l'Energie Atomique (Saclay, France) fellowship.

** To whom correspondence should be addressed. Tel.: 331-69088244; Fax: 331-69084712; E-mail: toledano@jonas.saclay.cea.fr.

Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009814200


    ABBREVIATIONS

The abbreviations used are: YPD, yeast extract, peptone, and glucose; SM, synthetic medium; SD, minimal medium.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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