The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae

In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isoform (peroxisomal catalase), the mutant showed the same levels of total catalase activity as the control strain. Furthermore, in cells deficient in Ctt1, the reduced glutathione:oxidized glutathione ratio (GSH:GSSG) of dry cells was higher than that of the control strain, indicating a compensatory mechanism of defense in response to dehydration. Even so, desiccation tolerance of the ctt1 strain was significantly lower than in the control strain. Using a fluorescent probe sensitive to oxidation, we observed that cells of the ctt1 strain showed levels of intracellular oxidation 70% higher than those of control strain, suggesting that Ctt1 plays a role in the maintenance of the intracellular redox balance during dehydration and, therefore, in tolerance against a water stress.     

The effect of superoxide dismutase deficiency on cadmium stress

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.     

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.     

Oxidative stress resistance of Escherichia coli strains deficient in glutathione biosynthesis

Sensitivity to various oxidants was determined for Escherichia coli strains JTG10 and 821 deficient in biosynthesis of glutathione (gsh-) and their common parental strain AB1157 (gsh+). The three strains showed identical sensitivity to H2O2. E. coli 821 was more resistant than AB1157 and JTG10 to menadione, cumene hydroperoxide, and N-ethylmaleimide. This resistance was not related to the gsh mutation because the other gsh- mutant and the parental strain showed similar sensitivity to these oxidants. The measured activities of NADPH:menadione diaphorase and glucose-6-phosphate dehydrogenase and the extracellular level of menadione suggested that the enhanced resistance of E. coli 821 to menadione might be due to decreased diaphorase activity, but not to a lowered rate of menadione uptake.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the methylotrophic yeast Hansenula polymorpha

The Hansenula polymorpha GSH1/MET1 gene was cloned by complementation of glutathione-dependent growth of H. polymorpha gsh1 mutant isolated previously as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resistant and cadmium ion sensitive clone. The H. polymorpha GSH1 gene was capable of restoring cadmium ion resistance, MNNG sensitivity, normal glutathione level and cell proliferation on minimal media without addition of cysteine or glutathione, when introduced into the gsh1 mutant cells. It was shown that the H. polymorpha GSH1 gene has homology to the Saccharomyces cerevisiae MET1 gene encoding S-adenosyl-L-methionine uroporphyrinogen III transmethylase, responsible for the biosynthesis of sulfite reductase cofactor, sirohaem. The H. polymorpha GSH1/MET1 gene deletion cassette (Hpgsh1/met1::ScLEU2) was constructed and corresponding null mutants were isolated. Crossing data of the point gsh1 and null gsh1/met1 mutants demonstrated that both alleles were located to the same gene. The null gsh1/met1 mutant showed total growth restoration on minimal media supplemented with cysteine or glutathione as a sole sulfur source, but not with inorganic (sulfate, sulfite) or organic (methionine, S-adenosylmethionine) sources of sulfur. Moreover, both the point gsh1 and null gsh1/met1 mutants displayed increased sensitivity to the toxic carbon substrate methanol, formaldehyde, organic peroxide and cadmium ions.     

Cloning of the cDNA and genomic clones for glutathione synthetase fromArabidopsis thaliana and complementation of agsh2 mutant in fission yeast

Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated fromArabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast,Schizosaccharomyces pombe, and shared only a small region of similarity with theEscherichia coli protein. A 4.3 kbSstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons.When theArabidopsis cDNA cloned in a special shuttle vector was expressed in aS. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in thegsh2 ^- mutant, and restored to substantial levels by the expression of theArabidopsis cDNA. TheS. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols,¹⁰⁹Cd²⁺ binding activity, and cadmium resistance. Since theArabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.     

The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain

A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.     

Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.     

一种适用于产朊假丝酵母的基因敲除系统及其在gsh1基因敲除中的应用

报道一种适用于产朊假丝酵母Candida utilis的基因敲除系统,利用该敲除系统获得gsh1基因敲除杂合突变株。根据不同种属酵母菌γ-谷氨酰半胱氨酸合成酶(γ-GCS)蛋白质的保守序列,克隆C.utilis SZU 07-01的gsh1基因;以商品化质粒pPICZalpha A为基础,构建gsh1基因的敲除载体pPICZalpha A-kan 3,其中,kan基因的启动子TEF被替换为来自于C.utilis SZU 07-01的GAP启动子(pGAP:kan)。质粒电转化C.utilis,获得gsh1基因敲除杂合突变株C.utilis GSH-6。结合发酵培养得到的数据进行分析,突变株的γ-GCS酶活比出发菌株降低17.5%,GSH合成量降低61%,细胞干重降低18.5%。所构建敲除组件pGAP:kan的成功应用为从分子水平研究C.utilis中谷胱甘肽(GSH)的生理功能提供了一种新借鉴。     

Co-amplification of the gamma-glutamylcysteine synthetase gene gsh1 and of the ABC transporter gene pgpA in arsenite-resistant Leishmania tarentolae.

Resistance to the oxyanion arsenite in the parasite Leishmania is multifactorial. We have described previously the frequent amplification of the ABC transporter gene pgpA, the presence of a non-PgpA thiol-metal efflux pump and increased levels of glutathione and trypanothione in resistant cells. Other loci are also amplified, although their role in resistance is unknown. By gene transfection, we have characterized one of these novel genes. It corresponds to gsh1, which encodes gamma-glutamylcysteine synthetase, an enzyme involved in the rate-limiting step of glutathione biosynthesis. Transfection of gsh1 in wild-type cells increased the levels of glutathione and trypanothione to levels found in resistant mutants. These transfectants were not resistant to metals. However, when gsh1 was transfected in partial revertants, it conferred resistance. As pgpA is frequently co-amplified with gsh1, we co-transfected the two genes into both wild-type and partial revertants. Arsenite resistance levels in wild-type cells could be accounted for by the contribution of PgpA alone. In the partial revertant, the gsh1 and pgpA gene product acted synergistically. These results support our previous suggestion that PgpA recognizes metals conjugated to thiols. Furthermore, amplification of gsh1 overcomes the rate-limiting step in the synthesis of trypanothione, contributing to resistance. In addition, the results suggest that at least one more factor acts synergistically with the gsh1 gene product.     

Protection against oxidation during dehydration of yeast

Based on the well-documented notion that oxygen affects the stability of dried cells, the role of the cytosolic and mitochondrial forms of superoxide dismutase (Sod) in the capacity of cells to resist dehydration was examined. Both enzymes are important for improving survival, and the absence of only 1 isoform did not impair tolerance against dehydration. In addition, sod strains showed the same Sod activity as the control strain, indicating that the deficiency in either cytoplasmic Cu/Zn or mitochondrial Mn was overcome by an increase in activity of the remaining Sod. To measure the level of intracellular oxidation produced by dehydration, a fluorescent probe, 2',7'-dichlorofluorescein, was used. Dry cells exhibited a high increase in fluorescence: both control and sod mutant strains became almost 10-fold more oxidized after dehydration. Furthermore, the disaccharide trehalose was shown to protect dry cells against oxidation.     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Redox regulation of the tumor suppressor PTEN by glutathione

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Δ and gsh2Δ mutants. Expression of γ-glutamylcysteine synthetase Gsh1 in the gsh1Δ mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.     

Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in gamma-l-glutamyl-l-cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, gamma-glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that gamma-glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4-dependent expression of GSH1 and elevating the cellular levels of gamma-glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4-dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4-regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1-specific mechanisms exist that respond to GSH availability.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

Molecular cloning of Drosophila gamma-glutamylcysteine synthetase by functional complementation of a yeast mutant

gamma-Glutamylcysteine synthetase (GCS) catalyses a critical, rate-limiting step in glutathione synthesis. In this study we describe the isolation and characterisation of a GCS cDNA (pDmGCS4.3. 3) from Drosophila melanogaster by functional complementation of a Saccharomyces cerevisiae gsh1 mutant. Expression of pDmGCS4.3.3 in the yeast mutant partially restored glutathione levels and conferred resistance to methylglyoxal. The pDmGCS4.3.3 cDNA was found to be approx. 4.6 kb in length, containing a 2 kb fragment encoding an open reading frame with a high degree of deduced amino acid sequence identity with previously reported GCS sequences. In situ hybridisation revealed that the Drosophila GCS gene maps to 7D6-9 on the X chromosome.