A Genetic Investigation of the Essential Role of Glutathione

Abstract

In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of ⌬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 ⌬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 ␥-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 ⌬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. Glutathione (GSH) is a broadly conserved tripeptide with a highly reactive thiol and a very low redox potential of about Ϫ240 to Ϫ250 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 GSHcontaining organisms, GSH is synthesized in two steps beginning with the action of ␥-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 ␥-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 ␥-glutamyl cysteine synthetase ...

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

“…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.…”

“…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.…”

“…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 Saccharomyces cerevisiae Proteome of Oxidized Protein Thiols

“…The Oxidized Proteome of Cells with an Altered Glutathione Pathway-The GSH pathway was tested with the ⌬gsh1PRO2-1⌬glr1 strain (22), which carries deletions of both GLR1 (encoding glutathione reductase) and GSH1 (encoding ␥-glutamylcysteine synthase, the rate-limiting enzyme in GSH biosynthesis) and which harbors a third mutation in the proline biosynthetic enzyme gene PRO2, which restores the synthesis of GSH to Ͻ0.5% of the wild-type cell content. These cells did not show significant variations in protein levels compared with wild-type cells (data not shown).…”

“…(21), and isogenic derivatives. The ⌬gsh1PRO2-1⌬glr1 (yeast SOG⌬glr1) strain has been described (22). Chromosomal gene inactivation was performed by oligonucleotide-mediated PCR replacement of entire coding regions and confirmed by PCR.…”