Oxidative Stress In Yeast Cells Research Articles

Identification of Whirly transcription factors in Triticeae species and functional analysis of TaWHY1-7D in response to osmotic stress.

Osmotic stress poses a threat to the production and quality of crops. Whirly transcription factors have been investigated to enhance stress tolerance. In this study, a total of 18 Whirly genes were identified from six Triticeae species, which were classified into Whirly1 and Whirly2. The exon-intron structure, conserved motif, chromosomal location, collinearity, and regulatory network of Whirly genes were also analyzed. Real-time PCR results indicated that TaWHY1 genes exhibited higher expression levels in leaf sheaths and leaves during the seedling stage, while TaWHY2 genes were predominantly expressed in roots. Under PEG stress, the expression levels of TaWHY1-7A, TaWHY2-6A, TaWHY2-6B, and TaWHY2-6D were increased, TaWHY1-7D was reduced, and TaWHY1-4A had no significant change. All TaWHY genes were significantly up-regulated in response to NaCl stress treatment. In addition, TaWHY1-7A and TaWHY1-7D mainly enhanced the tolerance to oxidative stress in yeast cells. TaWHY2s mainly improved NaCl stress tolerance and were sensitive to oxidative stress in yeast cells. All TaWHYs slightly improved the yeast tolerance to d-sorbitol stress. The heterologous expression of TaWHY1-7D greatly improved drought and salt tolerance in transgenic Arabidopsis. In conclusion, these results provide the foundation for further functional study of Whirly genes aimed at improving osmotic stress tolerance in wheat.

The Reaction of the Yeast Saccharomyces cerevisiae to Contamination of the Medium with Aflatoxins B2 and G1, Ochratoxin A and Zearalenone in Aerobic Cultures.

The mechanisms by which yeast cells respond to environmental stress include the production of heat shock proteins (HSPs) and the reduction of oxidative stress. The response of yeast exposed to aflatoxins B2+G1 (AFB2+G1), ochratoxin A (OTA), and zearalenone (ZEA) in aerobic conditions was studied. After 72 h of yeast cultivation in media contaminated with mycotoxins, the growth of yeast biomass, the level of malondialdehyde, and the activity of superoxide dismutase, glutathione S-transferase and glutathione peroxidase were examined; the expression profile of the following heat shock proteins was also determined: HSP31, HSP40, HSP60, HSP70, and HSP104. It was demonstrated that at the tested concentrations, both AFB2+G1 and ZEA inhibited yeast biomass growth. OTA at a concentration of 8.4 [µg/L] raised the MDA level. Intensified lipoperoxidation and increased activity of SOD and GPx were observed, regardless of the level of contamination with ZEA (300 µg/L or 900 µg/L). Increased contamination with AFB2+G1 and OTA caused an increase in the production of most HSPs tested (HSP31, HSP40, HSP70, HSP104). ZEA contamination in the used concentration ranges reduced the production of HSP31. The response of yeast cells to the presence of mycotoxin as a stressor resulted in the expression of certain HSPs, but the response was not systematic, which was manifested in different profiles of protein expression depending on the mycotoxin used. The tested mycotoxins influenced the induction of oxidative stress in yeast cells to varying degrees, which resulted in the activation of mainly SOD without GST mobilization or with a small involvement of GPx.

Antioxidant activity of fruit jellies enriched with phytochemicals from Pinus sylvestris L.

The aim of this study was to examine the antioxidant properties of fruit jellies made from concentrated fruit juice with the addition of bioactive compounds acquired from young pine shoots. The scope of work included: determination of the optimal extraction temperature of bioactive compounds with the use of concentrated apple juice as an osmotic agent, preparation of jellies from obtained extract, as well as analysis of the phytochemical and antioxidant activity of jellies using S. cerevisiae as a model organism. The study showed that concentrated apple juice can be a good extractant of bioactive ingredients contained in young pine shoots. However, it has been demonstrated that the recovery efficiency of these components is dependent on the process temperature. The most efficient extraction of phytochemicals can be achieved by conducting the process at 60 °C. The jellies made from the extract showed high antioxidant activity which was closely correlated with the amount of raw material used. In addition, the composition of bioactive compounds presented in the jellies reduced the level of oxidative stress in yeast cells treated with H2O2, which was demonstrated by lower intracellular ROS and lower SOD activity. The result was reduced cytotoxicity of hydrogen peroxide and increased cell viability.

Expression of the human antiapoptotic protein Bcl-2 increases nerolidol production in engineered yeast

Mammalian B-cell lymphoma/leukemia 2 (Bcl-2) has cytoprotective effects against oxidative stress in yeast cells. This study showed that human Bcl-2 increased nerolidol production in an engineered Saccharomyces cerevisiae strain with higher cell viability and lower reactive oxygen species levels. Our previous study found that the overexpression of HAC1, the key regulator of endoplasmic reticulum stress, increased the nerolidol titer levels. Endoplasmic reticulum stress has crosstalk with oxidative stress. We thus investigated the coincident effect of the overexpression of BCL-2 and HAC1, and found that both of them changed the transcription of genes involved in the fatty acid metabolism pathway and resulted in increased nerolidol synthesis. The addition of 3 mM linoleic acid further increased the nerolidol titer to 725.5 mg/L. Finally, fed-batch fermentation was implemented and resulted in the accumulation of 15.5 g/L nerolidol. This study provides a simple and effective strategy for sesquiterpene production in engineered yeast.

Roles of NOD1/Rip2 signal pathway in carotid artery remodelling in spontaneous hypertensive rats.

Nucleotide-binding and oligomerization domain (NOD) receptor is a member of inherent immunity recognition receptor family. We investigated the NOD1/Rip2 signalling pathway on carotid arterial remodelling in spontaneously hypertensive rats (SHRs). SHRs were treated with NOD1 agonist (iE-DAP), inhibitor (ML130), or normal saline. We determined the NOD1 and Rip2 expression in carotid artery tissues, serum tumour necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1). The carotid artery remodelling in 16-week SHRs was higher than that of 8-week SHRs and 16-week Wistar-Kyoto (WKY) rats. Expression of NOD1, Rip2, MCP-1 and TNF-α in 16-week SHRs was higher than that of 8-week SHRs and 16-week WKY rats. Blood pressure in iE-DAP-treated SHRs was higher than SHR-C group (no treatment), together with MCP-1, TNF-α, NOD1 and Rip2 expression, as well as carotid artery remodelling. In ML130-treated group, these aspects were completely the opposite. Taken together, inhibition of NOD1/Rip2 signalling pathway could delay the vascular remodelling process.

Multifunctional cationic surfactants with a labile amide linker as efficient antifungal agents-mechanisms of action.

Our research aimed to expand the knowledge of relationships between the structure of multifunctional cationic dicephalic surfactants with a labile linker-N,N-bis[3,3-(dimethylamine)propyl]alkylamide dihydrochlorides and N,N-bis[3,3-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9H19, n-C11H23, n-C13H27, n-C15H31)-and their possible mechanism of action on fungal cells using the model organism Saccharomyces cerevisiae. General studies performed on surfactants suggest that in most cases, their main mechanism of action is based on perforation of the cell membranes and cell disruption. Experiments carried out in this work with cationic dicephalic surfactants seem to modify our understanding of this issue. It was found that the investigated compounds did not cause perforation of the cell membrane and could only interact with it, increasing its permeability. The surfactants tested can probably penetrate inside the cells, causing numerous morphological changes, and contribute to disorders in the lipid metabolism of the cell resulting in the formation of lipid droplet aggregates. This research also showed that the compounds cause severe oxidative stress within the cells studied, including increased production of superoxide anion radicals and mitochondrial oxidative stress. Dicephalic cationic surfactants due to their biodegradability do not accumulate in the environment and in the future may be used as effective antifungal compounds in industry as well as medicine, which will be environmentally friendly. KEY POINTS: • Dicephalic cationic surfactants do not induce disruption of the cell membrane. • Surfactants could infiltrate into the cells and cause accumulation of lipids. • Surfactants could cause acute oxidative stress in yeast cells. • Compounds present multimodal mechanism of action. Graphical abstract.

Adriamycin inhibits glycolysis through downregulation of key enzymes in Saccharomyces cerevisiae.

Adriamycin is a widely used drug for the treatment of various types of cancers, but its clinical application is limited because of irreversible dilated cardiomyopathy. The incidence of cardiomyopathy is a consequence of disrupted energy production, which could be related to the defects in glycogen, lipid and mucopolysaccharide metabolism. We explored the effect of Adriamycin on enzymes involved in glycolysis and apoptotic genes through molecular docking. We used Saccharomyces cerevisiae as model organism and studied the effect of Adriamycin on selected enzymes involved in glycolysis. The docking studies revealed that Adriamycin interacts with phosphofructokinase and enolase in an efficient manner. In phosphofructokinase, Adriamycin binds at the active site and with enolase the drug interacts at the cofactor-binding site (Mg2+) which might impair the activity of the enzyme. Gene expression studies revealed that Adriamycin causes the dysregulation of glycolysis through dysregulation of hexokinase, phosphoglycerate mutase, enolase and downregulation of pyruvate kinase. The drug shows a biphasic effect on the expression of genes enolase and pyruvate kinase. The impairment in glycolysis might reduce the ATP synthesis, and the cells might be deprived of energy. The condition is further worsened by elevated ROS levels triggering the cell to undergo apoptosis evidenced by downregulation of SOD and upregulation of BAX and caspase. In conclusion, our study reveals that Adriamycin impairs glycolysis and cause cell to undergo apoptosis due to oxidative stress in yeast cells.

Yeast Chemogenetic Screening as a Tool to Unravel the Antifungal Mode of Action of Two Selected Selenocyanates

During recent decades, selenium-containing compounds, as with the chemically similar sulfur-containing compounds, have gained considerable interest as cytotoxic and anticancer agents. Selenocyanates represent a well-established class of organic selenium compounds. These agents exhibit a wide range of biological activities. Classically, selenocyanates may cause an increase in the intracellular levels of reactive oxygen species (ROS) and exert cytotoxic activities, thus, acting as pro-oxidants. In this study, chemogenetic profiling was carried out to decipher the resistance mechanisms as central part of the antifungal mode of action against two selected selenocyanates. If a mutant line is less resistant against a compound compared to the wildtype, the gene deleted in that strain seems to be correlated with the resistance. Yeast mutants carrying gene deletions for specific redox-related protein function were employed in the chemogenetic screening. The results of screening reveal the hypersensitivity of mutants carrying deletions for glutathione pool and metabolism. To confirm the results, Arabidopsis mutants deficient in glutathione were subjected to various concentrations of selenocyanates to observe their effects on mutants and the wildtype. A significant dose dependent inhibition in Arabidopsis mutants compared to the wildtype confirmed the findings of the chemogenetic screening. The data suggest that the two representatives of organoselenium compounds cause oxidative stress in yeast cells and glutathione participates towards the development of resistance against the chemicals.

The Truth of Toxicity Caused by Yttrium Oxide Nanoparticles to Yeast Cells.

Yttrium oxide (Y₂O₃) nanoparticles have widespread applications; however, toxicity due to these nanoparticles has also been reported. In this study, we evaluated the in vitro toxicity of Y₂O₃ nanoparticles according to the technical specifications published by the International Standard Organization (ISO/TS 19337:2016). We used Saccharomyces cerevisiae as a model microorganism represented the environment. We carried out catch ball analysis of yttrium oxide and yttrium ion toxicities. The result showed that Y₂O₃ nanoparticles (20 mg/5 ml) and YCl₃ (5 mg/5 ml) treatment caused oxidative stress in yeast cells. Based on transcriptome analysis, fluorescent spectroscopy, and solubility analysis of Y₂O₃ nanoparticles, we conclude that the toxicity is due to yttrium ions derived from the nanoparticles. The ions induce oxidative stress and cause protein denaturation, which in turn induces proteasome formation to eliminate denatured proteins. Yttrium nanoparticles induce oxidative stress, which has associated with heavy metal ions. Thus, the use of yttrium nanoparticles or yttrium ions must be controlled like heavy metals.

Xylene causes oxidative stress and pronounced translation repression in Saccharomyces cerevisiae

Organic solvent-resistant microorganisms are strongly desired for efficient fermentative production of hydrophobic substances in water-organic solvent two-phase systems. To improve organic solvent-resistance of microorganisms, a better understanding of the effects of organic solvents on microbial cells and cellular responses to organic solvents is essential. So far, various bacteria have been studied for their response mechanisms against organic solvents and improvement of their resistance to organic solvents. On the other hand, limited information is available on the effects of organic solvents on eukaryotic microorganisms. We herein examined the physiological effects of xylene, one of representative organic solvents, on the budding yeast Saccharomyces cerevisiae. We found that xylene induced fragmentation of mitochondria and the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, followed by the transcriptional activation of its target genes, GPX2 and TRX2, in yeast cells treated with xylene. These findings indicate that xylene caused oxidative stress in yeast cells. However, treatment with 0.03% (v/v) or more of xylene severely repressed the translation activity of yeast cells. Therefore, the expected protein synthesis of Yap1-target genes was not observed despite the transcriptional activation in cells treated with 0.03% (v/v) xylene. This is the first report on the inhibitory effects of xylene on bulk translation activity and provides novel insights into the toxicity of xylene.

The Effect of Berry Juices on the Level of Oxidative Stress in Yeast Cells Exposed to Acrylamide

Many food products, especially highly processed, contain numerous toxic substances, one of which is acrylamide. The mechanism of its toxicity has been associated with oxidative stress. The aim of this study was to investigate the effect of berry juices (bilberry, black mulberry and raspberry) on the level of oxidative stress in Saccharomyces cerevisiae cells exposed to acrylamide. Our results show that berry juices significantly protect yeast cells from negative effects of acrylamide. The protective effects of berries were manifested by restoring the growth of yeast cells and decreasing both the level of reactive oxygen species and protein carbonyl groups. Furthermore, bilberry juice shows the most effective protection, probably due to the optimal content of antioxidants. Practical Applications As natural food ingredients, fruits are an important source of substances with antioxidant properties. In particular, berries with intense dark skin color, such as blueberries, mulberries and raspberries, are the primary object of interest by nutritionists and consumers all over the world. These fruits are not only a source of vitamins and trace elements which are necessary for human health, but also of antioxidants such as polyphenolic compounds. Polyphenols play an important role in the prevention of obesity, diabetes and cardiovascular diseases; they are also known for their anti-cancer and anti-aging action. Looking for new opportunities to use berry fruits, we decided to test their ability to protect the organism against the toxic effect of ingredients present in highly processed foodstuffs. Our results show that berry juices may provide effective protection against toxicity of acrylamide. These results are further evidence of the benefits arising from the consumption of fruits.

Dimethyl sulfoxide induces oxidative stress in the yeastSaccharomyces cerevisiae

Dimethyl sulfoxide (DMSO) is used as a cryoprotectant for the preservation of cells, including yeast, and as a solvent for chemical compounds. We report that DMSO induces oxidative stress in the yeast. Saccharomyces cerevisiae wt strain EG-103 and its mutants Δsod1, Δsod2, and Δsod1 Δsod2 were used. Yeast were subjected to the action of 1-14% DMSO for 1 h at 28 °C. DMSO induced a concentration-dependent inhibition of yeast growth, the effect being more pronounced for mutants devoid of SOD (especially Δsod1 Δsod2). Cell viability was compromised. DMSO-concentration-dependent activity loss of succinate dehydrogenase, a FeS enzyme sensitive to oxidative stress, was observed. DMSO enhanced formation of reactive oxygen species, estimated with dihydroethidine in a concentration-dependent manner, the effect being again more pronounced in mutants devoid of superoxide dismutases. The content of cellular glutathione was increased with increasing DMSO concentrations, which may represent a compensatory response. Membrane fluidity, estimated by fluorescence polarization of DPH, was decreased by DMSO. These results demonstrate that DMSO, although generally considered to be antioxidant, induces oxidative stress in yeast cells.

Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccharomyces cerevisiae

Vanillin and furfural are derived from lignocellulosic biomass and inhibit yeast growth and fermentation as biomass conversion inhibitors. Furfural has been shown to induce oxidative stress in Saccharomyces cerevisiae. Since there has been no report on the relationship between vanillin and oxidative stress, we investigated whether vanillin caused oxidative stress in yeast cells. We showed that vanillin caused the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, and subsequent transcriptional activation of Yap1-target genes. The growth of the null mutant of the YAP1 gene (yap1Δ) was delayed in the presence of vanillin, which indicated that Yap1 plays a role in the acquisition of tolerance to vanillin. We also demonstrated that vanillin facilitated the fragmentation of mitochondria. These findings suggest that the toxicity of vanillin involves damage induced by oxidative stress.

Expression of Ovotransferrin Enhances Tolerance of Yeast Cells toward Oxidative Stress

Recently, we found that ovotransferrin (OTf) undergoes distinct self-cleavage in a redox-dependent process and exhibited in vitro superoxide dismutase (SOD)-like activity. In this study, we explore that the expression of OTf confers high tolerance to oxidative stress in yeast cells. The OTf gene was cloned into the vector pPICZB and was successfully expressed in methylotrophic yeast, Pichia pastoris KM71H. There was no growth difference between the non-transformed strain and recombinant strains harboring a mock vector (pPICZB) or the OTf gene carrying a vector (OTf-pPICZB). Intracellularly expressed OTf was found to undergo self-cleavage, producing a major fragment of 15 kDa, which corresponded to the disulfide kringle domain of the N-terminal lobe. The yeast OTf transformants exhibited strong tolerance to oxidative stress induced by either hydrogen peroxide (H₂O₂) or diethyl maleate (DEM). Further, OTf transformants showed higher intracellular reducing capacity and enhanced cytosolic reductase activity. This study is the first to describe the ability of OTf to confer in vivo antioxidative stress function within a complicated milieu of eukaryotic cells and provide novel insights for the potential of the OTf gene for molecular breeding of industrial yeast strains with high tolerance to oxidative stress.

Glutathione reductase from Brassica rapa affects tolerance and the redox state but not fermentation ability in response to oxidative stress in genetically modified Saccharomyces cerevisiae

To determine whether the exogenous expression of glutathione reductase (GR) from Brassica rapa subsp. pekinensis (BrGR) can reduce the deleterious effects of unfavorable conditions, we constructed a transgenic Saccharomyces cerevisiae strain bearing the GR gene cloned into the yeast expression vector, pVTU260. BrGR expression was confirmed by semi reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, immunoblotting analysis and an enzyme assay. Ectopic BrGR-expression improved cellular glutathione (GSH) homeostasis after higher GSH accumulation in the transgenic yeast than in the wild-type yeast under H(2)O(2)-induced oxidative stress. The BrGR-expressing yeast strain induced the activation of metabolic enzymes (Hxt, G6PDH, GAPDH and Ald), antioxidant systems (Gpx, Trx2, Trx3, Trr1, Tsa1 and porin) and molecular chaperones (Hsp104, Hsp90, Hsp70, Hsp42, Hsp26, Grp, Sti1 and Zpr1), which led to lower oxidative protein damage after a reduction in the level of cellular ROS in the BrGR-expressing yeast strain exposed to H(2)O(2) than in the wild-type yeast strain. BrGR-expression increased the ability to adapt and recover from H(2)O(2)-induced oxidative stress and various stressors, including heat shock, menadione, tert-butyl hydroperoxide, heavy metals, sodium dodecyl sulfate, ethanol and NaCl, but did not affect fermentation capacity. These results suggest that ectopic BrGR expression confers acquired tolerance by improving proteostasis and redox homeostasis through co-activation of various cell rescue proteins against ROS-induced oxidative stress in yeast cells.

Ascorbate and thiol antioxidants abolish sensitivity of yeast Saccharomyces cerevisiae to disulfiram

Sensitivity of baker’s yeast to disulfiram (DSF) and hypersensitivity of a mutant devoid of Cu, Zn-superoxide dismutase to this compound is reported, demonstrating that yeast may be a simple convenient eukaryotic model to study the mechanism of DSF toxicity. DSF was found to induce oxidative stress in yeast cells demonstrated by increased superoxide production and decrease of cellular glutathione content. Anoxic atmosphere and hydrophilic antioxidants (ascorbate, glutathione, dithiothreitol, cysteine, and N-acetylcysteine) ameliorated DSF toxicity to yeast indicating that oxidative stress plays a critical role in the cellular action of DSF.

Buffer modulation of menadione-induced oxidative stress in Saccharomyces cerevisiae

The objective of this study was to compare, in vivo, the effects of bicarbonate and phosphate buffers on survival and menadione-induced oxidative stress in yeast cells. The latter were treated with different concentrations of menadione in the presence of these two buffers. At 25 mM concentration of buffers, menadione only slightly reduced yeast surviving; at 50 mM concentration, cell killing by menadione was much more pronounced in bicarbonate than in phosphate buffer. Although the content of protein carbonyl groups did not show development of oxidative stress under menadione-induced stress, inactivation of aconitase and decrease in glutathione level mirrored its induction. However, cellular glutathione and aconitase activity decrease did not correlate with yeast survival. In vitro, aconitase was more quickly inactivated in 50 mM carbonate, than in 50 mM phosphate buffer. The possible involvement of the carbonate radical in these processes is discussed.

Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray

BackgroundMycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity.ResultsCitrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes – AADs, FLR1, OYE3, GRE2, and MET17 – were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair.ConclusionResults from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross hybridization.

Yeast cys3 and gsh1 mutant cells display overlapping but non-identical symptoms of oxidative stress with regard to subcellular protein localization and CDP-DAG metabolism.

In a screen for temperature-sensitive (37 degrees C) mutants of Saccharomyces cerevisiae that are defective in the proper localization of the Golgi transmembrane protein Emp47p, we uncovered a constitutive loss-of-function mutation in CYS3/STR1, the gene coding for cystathionine-gamma-lyase. We showed by immunofluorescence, sucrose-gradient analysis and quantitative Western analysis that the mutant mislocalized Emp47p to the vacuole at high temperature, while Golgi structures were apparently normal and biosynthetic routing of the vacuolar carboxypeptidase Y (CPY) and the plasma membrane GPI-anchored protein Gas1p were unaffected. The effect of high temperature on Emp47p localization, as well as the temperature sensitivity of the mutant strain on rich medium, appear to be caused by oxidative stress and are correlated with severe reductions in the intracellular levels of low-molecular-weight thiols. In accordance with this conclusion, cys3-2 mutant cells were more sensitive to the oxidizing agent 1-chloro-2,4-dinitrobenzene, which also aggravated the mislocalization of Emp47p observed at high temperature. Furthermore, all the phenotypes of the mutant were completely complemented by exogenous supply of the main low-molecular-weight thiol, glutathione (GSH) and, importantly, the thiol beta-mercaptoethanol reversed the temperature sensitivity of the mutant. A comparison of our mutant with a mutant defective in GSH synthesis showed that gsh1Delta cells were similar to wild-type cells under the stress conditions tested, with the exception of one novel oxidative stress-related phenotype that is observed in both cys3-2 and gsh1Delta mutant cells - a defect in CDP-DAG metabolism upon shift to the non-permissive temperature. As most of the stress-related phenotypes of cys3-2 mutant cells are more severe than those seen in gsh1Delta cells, we conclude that cysteine as such is required and sufficient to confer some degree of protection from oxidative stress in yeast cells.