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

Abstract

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...

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…Neither overexpression nor mRNA depletion of Grx1 affected proliferation of the parasites. A Grx1 null mutant of E. coli as well as the double mutant of Grx1 and Grx2 in yeast cells also did not show any proliferation defect under normal culture conditions (5,52). In the case of Grx2, RNAi resulted in growth retardation of procyclic T. brucei, which was the first indication that the two Grxs have non-redundant physiological functions.…”

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide

“…Furthermore, the Candida genome database (candidagenome.org/) currently describes four Grx (GRX1, GRX2, GRX3 and GRX5) and two thioredoxins (TRX1 and TRX2) for this species, further reinforcing that resistance to diamide is related to a compensatory mechanism for other Grx (or even thioredoxins) in C. albicans because these other genes might be up-regulated to act as thiol oxidoreductases responsible for reducing the protein disulphides or GSH-protein mixed disulphides generated during diamide-induced oxidative stress. Conversely, Luikenhuis et al (1998) found different functions for defences against diamide in S. cerevisiae that were dependent on the growth phase. In exponential phase cells, the lack of both Grx resulted in a resistance to diamide, whereas in stationary phase cells, the deletion of GRX1 alone led to resistance, suggesting that a complexity of mechanisms for different Grx in C. albicans may exist.…”

Selenite-induced cell death in Saccharomyces cerevisiae: protective role of glutaredoxins

“…The dithiol GRXs Grx1p and Grx2p (CPYC active site) are mainly cytosolic, but a minor portion of Grx2p is located within mitochondria (Luikenhuis et al, 1998;Porras et al, 2006). Despite the proposed role of Grx1p and Grx2p as general thiol oxidoreductases, their absence causes only moderate hypersensitivity to superoxide (in the case of Grx1p) and to superoxide and hydroperoxide (in the case of Grx2p) (Eckers et al, 2009;Luikenhuis et al, 1998). This phenotype could be explained based on the activity of these GRXs as glutathione peroxidases in in vitro assays (Collinson et al, 2002).…”