Saccharomyces cerevisiae

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Introduction
The yeast Succharomyces cerevisiae has been shown to possess distinct inducible protective oxidative stress responses to both hydrogen peroxide and superoxide anion generators, such as menadione [l-51. Other yeast species, such as Cundidu albicans [6] and Schizosaccharomyces pombe [7,8], have also recently been shown to have inducible oxidative stress responses.
In addition to enzymatic defences such as catalase and superoxide dismutase, all cells possess non-enzymatic defence systems to protect their cellular constituents against free radicals and reactive oxygen species, and to maintain cellular redox state. The most important example of these is glutathione (GSH), a tripeptide y-L-glutamyl+cystinylglycine [9]. This molecule acts as a radical scavenger, with the redox-active sulfhydryl group reacting with oxidants to produce oxidised gluthathione (GSSG). Surprisingly, in Escherichia coli, glutathione appears to be a dispensible antioxidant [lo]. In the yeast Saccharomyces cerevisiae, mutants deficient in y-glutamylcysteine synthetase (gshl) have been isolated and the gene cloned [11,12]. Recently, we have examined the role of oxidants in regulating the expression of GSHl (encoding y-glutamylcysteine synthetase) in yeast, and have demonstrated that the steady-state level of the GSHI mRNA is increased by exposure of cells to oxidants, particularly by superoxide anion generating agents, such as menadione [13]. Basal expression of the GSHl gene has been shown to be, at least partially,  Comparison of the degree of resistance to HsOs shown between adapted and non-adapted exponential cultures of S150-2B, and DJY144 (Ag&).
Exponential phase cultures, growing aerobically at 30°C in SD medium supplemented with 1% (w/v) casamino acids to ODsss =0.15 (ca 2.5X lo6 cells per ml) were divided into two aliquots, treated for 1 h at 30°C with 0.4 mM Hz02 and a control which was not treated with any oxidant. The cells were then exposed to increasing levels of HzOz. Data represent the mean of three independent experiments and the standard error was routinely less than IO%, with the exception of the DJY144 1 mM HzOs point where the standard error was 50%. dependent upon the transcription factor Yap1 [13,14].
We wished to extend these studies and were interested in determining the role of glutathione in the protection of yeast against oxidative stress. This study shows that glutathione is an important antioxidant molecule in yeast and that y-glutamylcysteine synthetase deficient mutants still possess inducible HzOz-and superoxide anion-responsive adaptive stress responses.

Yeast strains and techniques
Standard yeast methods and growth media were as described by Rose et al. [15]. SD media were supplemented with 1% (w/v) casamino acids (Difco Ltd) or 40 l.tg/ml glutathione.

Plasmid construction
The gshl insertion mutation was constructed by amplifying an 1.68 kb fragment of the GSHI gene, which includes a portion of the coding sequence, from genomic DNA using the following oligonucleotide primers in a PCR reaction; 5'-TCCCTGTTTA-TAAACGGCGGC-3' and 5'-CACGGAATACGC-AGCGTTCTC-3'.
The amplified fragment (from nucleotide -215 to +1463) was cloned into pT7-Blue T (Novagen Ltd), then excised as an EcoRI-Hind111 fragment and ligated to the EcoRI-Hind111 digested pTZ19r (Pharmacia Ltd). The 0.83 kb BglII fragment from pFL35 [20], containing the TRPI gene, was inserted into the BarnHI site in the GSHI coding sequence, creating plasmid pDJ69. The positions of the PCR primers are given relative to the ATG codon.

Menadione and Hz02 adaptation experiments
To determine the degree of protection against cell killing afforded by menadione and Hz02 pretreatment, exponential cultures of yeast strains were grown aerobically in SD media supplemented with casamino acids [15] at 30°C in the presence or ab-

D. W.S. Stephen. D.J. Jamieson I FEMS Microbiology Letters 141 (1996) 207-212
sence of either Hz02 (0.1 mM) or menadione (0.5 mM) for 1 h. Cells were collected by centrifugation and washed once in distilled Hz0 prior to resuspension in an equal volume of fresh pre-warmed SD medium. 1 ml aliquots of these cells were then incubated for 1 h at 30°C with increasing levels of either plumbagin or H20s, after which time cells were washed once in distilled HsO, plated out at appropriate dilutions on YPD plates and incubated at 30°C.

Sensitivity of a gshl mutant towards oxidants
During our characterisation of the oxidative stress responses of the yeast S. cerevisiae we decided to investigate the role of glutathione as an antioxidant. This was prompted by work in E. coli which suggested that glutathione was a dispensible antioxidant molecule [lo]. To determine whether this was also the case for S. cerevisiae we constructed a yeast strain containing an insertion mutation in the GSHI gene, which encodes y-glutamylcysteine synthetase, the rate limiting enzyme in the biosynthetic pathway of glutathione [9]. To confirm that this strain (DJY144) was defective in y-glutamylcysteine synthetase activity extracts of both wild-type and mutant cells grown under a variety of different conditions were assayed to determine the level of glutathione in the mutant. The results confirm that DJY144 has extremely low levels of cellular glutathione (Table 1). The data also show that exposure of wild-type cells to oxidants, particularly menadione, results in only a modest depletion of the total glutathione levels of up to 27% compared to unstressed cells. We found that the gshl insertion mu- Table 1 Effect of oxidants on intracellular glutathione levels of the degree of resistance to plumbagin shown between wild-type (SlSO-2B) and adapted and nonadapted exponential cultures of DJY144 (AgrW). Exponential phase cultures, growing aerobically at 3O'C in SD medium supplemented with 1% (w/v) casamino acids to ODem =0.15 (ca. 2.5 X lo6 cells per ml) were divided into two aliquots, treated for 1 h at 30°C with 0.1 mM menadione and a control which was not treated with any oxidant. The cells were then exposed to increasing levels of plumbagin. Data represent the mean of three independent experiments and the standard error was routinely less than 10%. tant grew normally in YPD media and failed to grow in minimal medium; normal growth rates were restored by supplementation of the medium with glutathione or acid digested casein (data not shown).

Eflect of gshl mutation on the inducible Hz02 and 02' adaptive stress responses
We have shown that yeast possesses separate adaptive stress responses towards both Hz02 and superoxide anions [l]. Because of the important antioxidant role for glutathione in mammalian cells [9], we were interested in determining the effect that a gshl insertion mutation would have on the two oxidant adaptive stress responses of S. cerevisiae. Cultures of the gshl mutant and wild-type yeast were Cultures of the wild type (S150-2B) and the gshl mutant (DJY 144) were grown to early exponential phase ODsm -0.15 and exposed to HzOz (0.4 mM) or the superoxide anion generating compound menadione (1 mM) for 1 h. After this time extracts were prepared and assayed for glutathione. The results are presented as pm01 ghrtathione per lo6 cells. Values shown are from three independent cultures. grown aerobically to early exponential phase in YPD media and exposed to various concentrations of HsOs for 1 h, after which time the viability of the cells was determined. Surprisingly, despite the normal growth of the mutant in YPD medium, the gshl mutant was much more sensitive than the wild type to the levels of HsOs used, even when grown in YPD medium (data not shown).
To examine the presence of the HsOs-adaptive stress response cultures of DJY144 (gsh-) were grown in SD plus casamino acids to early exponential phase and treated with a sub-lethal dose of HsOs (0.1 mM HsOs) for 1 h to induce the adaptive response. Aliquots of the treated and untreated cells were then exposed to increasing concentrations of HsOs for a further hour, whereupon the cells were collected and viability determined.
These results demonstrate that exposure to sub-lethal levels of Hz02 still induces an adaptive response in the gshl mutant (Fig. 1).
In view of the effect that redox cyclers such as menadione have on the expression of the GSHI gene [13] we were particularly interested in determining the effect of the gshl insertion mutation on the superoxide sensitivity and the superoxide anion inducible stress response. To investigate this, exponentially growing cultures of DJY144 (gshl : : TRPI) were exposed to 0.5 mM menadione for 1 h (to induce the adaptive stress response) then treated with increasing concentrations of another redox-cycling agent, plumbagin.
We have previously determined that the toxicity of plumbagin (which is structurally similar to menadione) is due to the production of superoxide anions [4]. The results show that the gshZ deficient mutant is hypersensitive to plumbagin compared to the wild type (Fig. 2). Despite the hypersensitivity of the gshl mutant this strain is still capable of inducing an adaptive stress response to superoxide anions (Fig. 2).
Our results show that glutathione is an important antioxidant molecule in the yeast S. cerevisiae, with gsh insertion mutants being hypersensitive to oxidants, particularly HsOs, but also to the superoxide anion generators menadione and plumbagin. This is in sharp contrast to the results obtained with E. coli [lo] and is in keeping with the results obtained from work in higher eukaryotes [21], reinforcing the utility of S. cerevisiae as an ideal eukaryotic model system cultures of wild-type (S150-2B) and DJY144 (Agshl). Cells were grown aerobically in SD medium supplemented with 1% (w/v) casamino acids at 30°C to saturation to give stationary-phase cultures. Aliquots of cells were then treated with increasing levels of either HsOs (A) or plumbagin (B). Data represent the mean of two independent experiments and the standard error was routinely less than 10%.
to study the cellular responses to oxidative stress. The defects of the gshl insertion mutant can all be reversed by the addition of GSH to the growth medium. Interestingly, growth of the mutant in YPD or casamino acids supplemented minimal medium, without added glutathione, is normal yet the mutant still remains hypersensitive towards oxidants. This suggests that at least some of the functions of glutathione can be provided by peptides and or amino acids but not all.

Role of glutathione in conferring resistance towards oxidants seen in stationary-phase cultures
We have previously demonstrated that S. cerevisiae cells grown past the diauxic shift point are con-Downloaded from https://academic.oup.com/femsle/article-abstract/141/2-3/207/533175 by guest on 30 July 2018

Microbiology Letters 141 (1996) 207-212 211
siderably more resistant towards oxidants than exponentially growing cells [l]. We were interested to determine what role glutathione played in this phenotype. We therefore examined the resistance of stationary-phase cultures of DJY144 towards both H202 and the superoxide anion generator plumbagin. Cultures of wild type and the gshl mutant were grown aerobically in casamino acids supplemented minimal medium to saturation and aliquots exposed to oxidants. We found that the gshl mutation resulted in greater sensitivity towards both Hz02 and redox-cycling compounds compared to that observed for wild-type yeast (Fig. 3). However, despite this, stationary-phase cultures of the gshl mutant were still more resistant to both Hz02 and plumbagin than exponentially growing cultures, suggesting that glutathione level is not the only factor involved in conferring resistance towards oxidants in stationaryphase cells.
We initiated this study as we are interested in determining how cells sense and react towards oxidative stress and were curious to determine what role GSH played in sensing this stress. Given the relatively abundant nature of GSH in cells and the presence of redox-active sulfhydryl groups we postulated that either changes in GSH levels or in the ratio of GSH/GSSG could be sensed by the cell and used as a signal to induce the adaptive stress responses. The presence of the oxidant adaptive stress responses in the gshl deficient mutant (which contains no glutathione) implies that the signal for induction of both the peroxide and superoxide stress responses cannot be a glutathione conjugate of menadione or other compound, nor is it likely to be glutathione levels or the ratio of oxidised to reduced glutathione. However, as the levels of Hz02 resistance induced in the gshl mutant are considerably lower than that observed with the wild type it is possible that there exists a glutathione-regulated stress response in addition to a glutathione independent one. Further work is required to determine whether this is indeed the case.