Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Grant, Chris M.; MacIver, Fiona H.; Dawes, Ian W.

doi:10.1091/mbc.8.9.1699

Cited by 180 publications

(162 citation statements)

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“…GSH1 encodes ␥-glutamylcysteine synthase the enzyme that catalyzes the first and rate-limiting step in glutathione biosynthesis (Grant et al, 1997). Increased Gsh1 expression should protect cells from cadmium stress because glutathione is important for cadmium detoxification (Perego and Howell, 1997).…”

Section: Cdc34/scf Met30 Is Required For the Cellular Response To Cadmentioning

confidence: 99%

“…To test whether Cdc34/SCF Met30 is involved in cadmium stress response, we tested growth of strains carrying temperature-sensitive mutations in components of SCF Met30 on cadmium-containing plates ( The cadmium sensitivity of met30 mutants was unexpected, because microarray experiments showed increased GSH1 expression in met30 mutants (our unpublished data). GSH1 encodes ␥-glutamylcysteine synthase the enzyme that catalyzes the first and rate-limiting step in glutathione biosynthesis (Grant et al, 1997). Increased Gsh1 expression should protect cells from cadmium stress because glutathione is important for cadmium detoxification (Perego and Howell, 1997).…”

Section: Cdc34/scf Met30 Is Required For the Cellular Response To Cadmentioning

confidence: 99%

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The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response

Yen

Kaiser

2005

MBoC

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Cells have developed a variety of mechanisms to respond to heavy metal exposure. Here, we show that the yeast ubiquitin ligase SCF Met30 plays a central role in the response to two of the most toxic environmental heavy metal contaminants, namely, cadmium and arsenic. SCF Met30 inactivates the transcription factor Met4 by proteolysis-independent polyubiquitination. Exposure of yeast cells to heavy metals led to activation of Met4 as indicated by a complete loss of ubiquitinated Met4 species. The association of Met30 with Skp1 but not with its substrate Met4 was inhibited in cells treated with cadmium. Cadmium-activated Met4 induced glutathione biosynthesis as well as genes involved in sulfuramino acid synthesis. Met4 activation was important for the cellular response to cadmium because mutations in various components of the Met4-transcription complex were hypersensitive to cadmium. In addition, cell cycle analyses revealed that cadmium induced a delay in the transition from G 1 to S phase of the cell cycle and slow progression through S phase. Both cadmium and arsenic induced phosphorylation of the cell cycle checkpoint protein Rad53. Genetic analyses demonstrated a complex effect of cadmium on cell cycle regulation that might be important to safeguard cellular and genetic integrity when cells are exposed to heavy metals. INTRODUCTIONHeavy metals are a major environmental hazard and present a danger to human health. The cause of the cytotoxic effects of heavy metals is not completely understood, but it has been suggested that at least part of their toxicity is due to the formation of hydroxyl radicals, which lead to lipid, protein, and DNA damage (Stohs and Bagchi, 1995;Brennan and Schiestl, 1996;Halliwell and Gutteridge, 1984).As with any cytotoxic and genotoxic insults, all organisms have developed strategies to respond to heavy metal exposure to maintain cellular and genetic integrity. These strategies include detoxification, repair, or removal of damaged molecules, and delay of cell division to prevent propagation of damaged cellular components (Jamieson, 1998).The biological effects of cadmium are perhaps better studied than that of other heavy metals. High affinity for sulfhydryl groups, competition with Zn(II) in proteins, nonspecific interaction with DNA, generation of reactive oxygen species, and depletion of glutathione have been shown to contribute to the toxicity of cadmium (Stohs and Bagchi, 1995;Zalups and Ahmad, 2003;McMurray and Tainer, 2003). Recently, it has been demonstrated in yeast that the genotoxic effects of cadmium are indirect (Jin et al., 2003;McMurray and Tainer, 2003). Rather than by direct DNA damage, cadmium leads to genome instability by inhibition of the DNA mismatch repair system (Jin et al., 2003). Although the mechanism of how cadmium inhibits DNA repair is not clear, it has been suggested that damage of sulfhydryl groups containing components of the mismatch repair system might be responsible (Jin et al., 2003).The damaging effect of cadmium on sulfhydryl groups containing pro...

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Section: Cdc34/scf Met30 Is Required For the Cellular Response To Cadmentioning

confidence: 99%

Section: Cdc34/scf Met30 Is Required For the Cellular Response To Cadmentioning

confidence: 99%

The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response

Yen

Kaiser

2005

MBoC

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“…Sensitivity to H 2 O 2 , tert-butyl hydroperoxide, cumene hydroperoxide, menadione, and diamide was determined by spotting strains onto YEPD plates containing various concentrations of oxidants (Grant et al, 1997). Cells were grown to stationary phase in YEPD, and 10-l aliquots of each culture, diluted to an A 600 of 3.0 and 0.3, were spotted onto appropriate plates.…”

Section: Sensitivity To Oxidantsmentioning

confidence: 99%

“…In addition, the grx1 grx2 double mutant was sensitive to tert-butyl hydroperoxide during both exponential and stationary phase growth (Figure 4). Diamide is a thiol-specific oxidant that can readily oxidize GSH (Kosower and Kosower, 1995), and stationary phase cells that lack TRX2, GSH1 or GSH2 show increased resistance to this oxidant (Muller, 1996;Grant et al, 1997). Similarly, the grx1 mutant was more resistant to 1.5 mM diamide than the wild-type strain during stationary phase.…”

Section: Strains Lacking Grx1 or Grx2 Are Sensitive To Conditions Of mentioning

confidence: 99%

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Luikenhuis

Perrone

Dawes

et al. 1998

MBoC

219

217

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Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40 -52% identity and 61-76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using ␤-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins. INTRODUCTIONGlutaredoxin from Escherichia coli was first discovered as a small, heat-stable protein required for the glutathione-dependent synthesis of deoxyribonucleotides catalyzed by ribonucleotide reductase (Holmgren, 1976). Glutaredoxin 1 is a 9-kDa protein that acts as a reduced glutathione (GSH)-dependent disulfide oxidoreductase by virtue of the two cysteine residues in its active site (Holmgren and Aslund, 1995). Later studies in mutants that lacked both glutaredoxin and thioredoxin revealed that E. coli actually contains three glutaredoxins (Grx1-3), with glutaredoxin 3 also able to function in ribonucleotide synthesis (Aslund et al., 1994). In contrast, glutaredoxin 2 was proposed to be the first member of a novel class of glutaredoxins that lack activity as hydrogen donors for ribonucleotide reductase (Vlamis-Gardikas et al., 1997). Glutaredoxins have subsequently been identified and isolated from various eukaryotes, including human, bovine, pig, yeast, and rice (Minakuchi et al., 1994;Holmgren and Aslund, 1995). The structure of these proteins has been highly conserved throughout evolution, particularly in the region of the active site (Wells et al., 1993;Holmgren and Aslund, 1995). However, despite extensive structural analysis, little is known regarding the biochemical function of these eukaryotic glutaredoxins in vivo.There appears to be considerable functional overlap between the glutaredoxin and thioredoxin systems. Similar to glutaredoxin, thioredoxin is a small, heatstable pro...

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“…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.…”

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confidence: 99%

A Genetic Investigation of the Essential Role of Glutathione

Spector

Labarre

Toledano

2001

Journal of Biological Chemistry

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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 ...

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Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Cited by 180 publications

References 45 publications

The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response

The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

A Genetic Investigation of the Essential Role of Glutathione

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Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

Cited by 180 publications

References 45 publications

The Yeast Ubiquitin Ligase SCFMet30Regulates Heavy Metal Response

The Yeast Ubiquitin Ligase SCFMet30Regulates Heavy Metal Response

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

A Genetic Investigation of the Essential Role of Glutathione

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The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response

The Yeast Ubiquitin Ligase SCF^Met30Regulates Heavy Metal Response