Gsh1 Mutants Research Articles

S-allylmercaptoglutathione Is a Substrate for Glutathione Reductase (E.C. 1.8.1.7) from Yeast (Saccharomyces cerevisiae).

Allicin (diallylthiosulfinate) is a potent thiol reagent and natural defense substance produced by garlic (Allium sativum) tissues when damaged. Allicin acts as a redox toxin and oxidizes the cellular glutathione (GSH) pool producing S-allylmercaptoglutathione (GSSA). The cellular enzyme glutathione reductase (GR) uses NADPH to reduce glutathione disulfide (GSSG) back to GSH and replenishes the GSH pool. It was not known whether GR could accept GSSA as a substrate. Here, we report that GR from yeast (Saccharomyces cerevisiae) shows Michaelis–Menten kinetics with GSSA as substrate in vitro (Km = 0.50 mM), but that GSSA is not as good a substrate as GSSG (Km = 0.07 mM). Furthermore, cells unable to synthesize GSH because the γ-glutamylcysteine synthetase (GSH1) gene is deleted, cannot grow without GSH supplementation and we show that the auxotrophic requirement for GSH in Δgsh1 mutants can be met by GSSA in the growth medium, suggesting that GSSA can be reduced to GSH in vivo.

Study on the glutathione metabolism of the filamentous fungus Aspergillus nidulans.

Yeast protein sequence-based homology search for glutathione (GSH) metabolic enzymes and GSH transporters demonstrated that Aspergillus nidulans has a robust GSH uptake and metabolic system with several paralogous genes. In wet laboratory experiments, two key genes of GSH metabolism, gcsA, and glrA, encoding γ-l-glutamyl-l-cysteine synthetase and glutathione reductase, respectively, were deleted. The gene gcsA was essential, and the ΔgcsA mutant required GSH supplementation at considerably higher concentration than the Saccharomyces cerevisiae gsh1 mutant (8-10 mmol l-1 vs. 0.5 μmol l-1). In addition to some functions known previously, both genes were important in the germination of conidiospores, and both gene deletion strains required the addition of extra GSH to reach wild-type germination rates in liquid cultures. Nevertheless, the supplementation of cultures with 10 mmol l-1 GSH was toxic for the control and ΔglrA strains especially during vegetative growth, which should be considered in future development of high GSH-producer fungal strains. Importantly, the ΔglrA strain was characterized by increased sensitivity toward a wide spectrum of osmotic, cell wall integrity and antimycotic stress conditions in addition to previously reported temperature and oxidative stress sensitivities. These novel phenotypes underline the distinguished functions of GSH and GSH metabolic enzymes in the stress responses of fungi.

Comparative Studies of Tri- and Hexavalent Chromium Cytotoxicity and Their Effects on Oxidative State of Saccharomyces cerevisiae Cells

Chromium is a significant mutagen and carcinogen in environment. We compared the effects of tri- and hexavalent chromium on cytotoxicity and oxidative stress in yeast. Cell growth was inhibited by Cr(3+) or Cr(6+), and Cr(6+) significantly increased the lethal rate compared with Cr(3+). Both Cr(3+) and Cr(6+) can enter into the yeast cells. The percent of propidium iodide permeable cells treated with Cr(3+) is almost five times as that treated with the same concentration of Cr(6+). Levels of TBARS, O2 (-), and carbonyl protein were significantly increased in both Cr(6+)- and Cr(3+)-treated cells in a concentration- and time-dependent manner. Moreover, the accumulation of these stress markers in Cr(6+)-treated cells was over the Cr(3+)-treated ones. The decreased GSH level and increased activity of GPx were observed after 300μM Cr(6+)-exposure compared with the untreated control, whereas there was no other change of GSH content in cells treated with Cr(3+) even at very high concentration. Exposure to both Cr(3+) and Cr(6+) resulted in the decrease of activities of SOD and catalase. Furthermore, the effect of Cr(6+) is stronger than that of Cr(3+). Null mutation sensitivity assay demonstrated that the gsh1 mutant was sensitive to Cr(6+) other than Cr(3+), the apn1 mutant is more sensitive to Cr(6+) than Cr(3+), and the rad1 mutant is sensitive to both Cr(6+) and Cr(3+). Therefore, Cr(3+) can be concluded to inhibit cell growth probably due to the damage of plasma membrane integrality in yeast. Although both tri- and hexavalent chromium can induce cytotoxicity and oxidative stress, the action mode of Cr(3+) is different from that of Cr(6+), and serious membrane damage caused by Cr(3+) is not the direct consequence of the increase of lipid peroxidation.

Glutathione and tryptophan metabolites are key players in Arabidopsis nonhost resistance against Colletotrichum gloeosporioides

The nonhost resistance of Arabidopsis against hemibiotrophic fungi in the genus Colletotrichum consists of pre- and post-invasive immune responses. Previously, we reported EDR1 and PEN2 as important components of Arabidopsis pre-invasive resistance toward non-adapted Colletotrichum gloeosporioides (Cg). However, despite their defect in entry control pen2 and edr1 mutants terminated further growth of this pathogen by activating the post-invasive hypersensitive response (HR) accompanied by plant cell death. In the present study, we showed that γ-glutamylcysteine synthetase (GSH1), which is required for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to pre- and post-invasive non-host resistance against Cg. We found GSH1 to be involved in the PEN2-dependent entry control of Cg. Opposite to pen2 and edr1, gsh1 mutants failed to restrict the invasive growth of the pathogen, which demonstrated the requirement for GSH1 during post-invasive non-host resistance. Based on the infection and metabolic phenotypes of Arabidopsis mutants defective in Trp metabolism, we showed that the biosynthesis of Trp-derived phytochemicals is also essential for resistance to Cg during the post-invasive HR. By contrast, GSH1 and these metabolites are dispensable for the induction of HR cell death, which is triggered in the non-invaded mesophyll cells adjacent to the Cg-invaded epidermal cells.

Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance

The tripeptide glutathione (GSH) is an important metabolite with a broad spectrum of functions, and its homeostasis is essential to maintain cellular redox poise and effective responses to stress in plants. In Arabidopsis GSH is synthesised in two successive enzymatic steps by γ-glutamylcysteine synthetase (GSH1), localised exclusively in plastids, forming the pathway intermediate γ-glutamylcysteine (γ-EC), and then by glutathione synthetase (GSH2), which is located in both plastids and cytosol. This suggests a mechanism for γ-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is speculated that the cytosol may be the main compartment for GSH biosynthesis. With the availability of knockout lethal mutants of GSH1 and GSH2 in Arabidopsis, we were able to manipulate the GSH biosynthetic pathway within cells through transgenic techniques. We successfully complemented the gsh1 and gsh2 null mutants with a cytosol-targeted bacterial EcGSHA and plastid-targeted Arabidopsis GSH2 protein, respectively, to wild-type phenotypes. These transgenics were little affected under heavy metal (cadmium) or oxidative stress (H2 O2 ) when compared to the wild type. Collectively, our data show that redirecting GSH1 activity exclusively to the cytosol or restricting GSH biosynthesis to the plastids has no significant impact on development or stress resistance, suggesting efficient exchange of γ-EC and GSH between the plastid and cytosol compartments within cells.

Development of glutathione-deficient embryos in Arabidopsis is influenced by the maternal level of glutathione

Glutathione (GSH) biosynthesis-deficient gsh1 and gsh2 null mutants of Arabidopsis thaliana have late embryonic-lethal and early seedling-lethal phenotypes, respectively, when segregating from a phenotypically wild-type parent plant, indicating that GSH is required for seed maturation and during germination. In this study, we show that gsh2 embryos generated in a partially GSH-deficient parent plant, homozygous for either the cad2 mutation in the GSH1 gene or homozygous for mutations in CLT1, CLT2 and CLT3 encoding plastid thiol transporters, abort early in embryogenesis. In contrast, individuals homozygous for the same combinations of mutations but segregating from heterozygous, phenotypically wild-type parents exhibit the parental gsh2 seedling-lethal phenotype. Similarly, homozygous gsh1 embryos generated in a gsh1/cad2 partially GSH-deficient parent plant abort early in development. These observations indicate that the development of gsh1 and gsh2 embryos to a late stage is dependent on the level of GSH in the maternal plant.

The γ‐glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants

Gamma-glutamylcysteine synthetase, encoded by the GSH1 gene, is the rate-limiting enzyme in the biosynthesis of glutathione and of trypanothione in Leishmania. The importance of GSH1 was assessed by generating GSH1 null mutants in Leishmania infantum. Removal of even a single wild-type allelic copy of GSH1 invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. However, by first supplementing the parasites with a rescue plasmid, we succeeded in obtaining both a single and null chromosomal GSH1 mutants. Parasites with one intact GSH1 chromosomal allele lost the rescuing plasmid but not the double knockout, when grown in the absence of antibiotic, indicating the essentiality of the GSH1 gene in Leishmania. Heterozygous mutants with one allele-inactivated transcribed less GSH1 mRNA and synthesized less glutathione and trypanothione. These mutants were more susceptible to oxidative stresses in vitro as promastigotes and showed decreased survival inside activated macrophages producing reactive oxygen or nitrogen species. These mutants showed a significant decreased survival in the presence of antimony (SbV) compared with control cells. All phenotypes were reverted in the add-back mutant, thus proving the importance of thiols in dealing with oxidants including the action of antimonials.

Distinct Temporal Requirements for the Homeobox Gene Gsx2 in Specifying Striatal and Olfactory Bulb Neuronal Fates

The homeobox gene Gsx2 (formerly Gsh2) is known to be required for striatal and olfactory bulb neurogenesis; however, its specific role in the specification of these two neuronal subtypes remains unclear. To address this, we have employed a temporally regulated gain-of-function approach in transgenic mice and found that misexpression of Gsx2 at early stages of telencephalic neurogenesis favors the specification of striatal projection neuron identity over that of olfactory bulb interneurons. In contrast, delayed activation of the Gsx2 transgene until later stages exclusively promotes olfactory bulb interneuron identity. In a complementary approach, we have conditionally inactivated Gsx2 in a temporally progressive manner. Unlike germline Gsx2 mutants, which exhibit severe alterations in both striatal and olfactory bulb neurogenesis at birth, the conditional mutants exhibited defects restricted to olfactory bulb interneurons. These results demonstrate that Gsx2 specifies striatal projection neuron and olfactory bulb interneuron identity at distinct time points during telencephalic neurogenesis.

Glutathione is necessary to ensure benefits of calorie restriction during ageing in Saccharomyces cerevisiae

Calorie restriction increases longevity of mammals and yeasts but this mechanism remains unclear. In this study, the role of glutathione on lifespan extension induced by calorie restriction was investigated by using a Saccharomyces cerevisiae strain deficient in glutathione synthesis ( gsh1). We observed an increase in chronological lifespan of calorie-restricted gsh1 mutant cells, compared to WT (wild type) strain, which was associated with a reduction in the levels of oxidative stress biomarkers. The gsh1 strain showed an increase in cell yield under calorie restriction that was associated with a higher pyruvate kinase activity and a reduction in oxygen consumption and aconitase activity. This indicates that the respiratory metabolism is decreased in gsh1 mutant cells. The lifespan extension of gsh1 mutant cells did not represent an advantage at long term, since old cells of gsh1 strain showed a higher frequency of petite mutants. In addition, aged WT cells outlast aged gsh1 mutant cells in direct competition assays in a fresh medium. These results suggest that glutathione is required for the beneficial effects of calorie restriction on cellular longevity.

Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells

Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

Identification of PAD2 as a γ‐glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis

The Arabidopsis pad2-1 mutant belongs to a series of non-allelic camalexin-deficient mutants. It was originally described as showing enhanced susceptibility to virulent strains of Pseudomonas syringae and was later shown to be hyper-susceptible to the oomycete pathogen Phytophthora brassicae (formerly P. porri). Surprisingly, in both pathosystems, the disease susceptibility of pad2-1 was not caused by the camalexin deficiency, suggesting additional roles of PAD2 in disease resistance. The susceptibility of pad2-1 to P. brassicae was used to map the mutation to the gene At4g23100, which encodes gamma-glutamylcysteine synthetase (gamma-ECS, GSH1). GSH1 catalyzes the first committed step of glutathione (GSH) biosynthesis. The pad2-1 mutation caused an S to N transition at amino acid position 298 close to the active center. The conclusion that PAD2 encodes GSH1 is supported by several lines of evidence: (i) pad2-1 mutants contained only about 22% of wild-type amounts of GSH, (ii) genetic complementation of pad2-1 with wild-type GSH1 cDNA restored GSH production, accumulation of camalexin in response to P. syringae and resistance to P. brassicae and P. syringae, (iii) another GSH1 mutant, cad2-1, showed pad2-like phenotypes, and (iv) feeding of GSH to excised leaves of pad2-1 restored camalexin production and resistance to P. brassicae. Inoculation of Col-0 with P. brassicae caused a coordinated increase in the transcript abundance of GSH1 and GSH2, the gene encoding the second enzyme in GSH biosynthesis, and resulted in enhanced foliar GSH accumulation. The pad2-1 mutant showed enhanced susceptibility to additional pathogens, suggesting an important general role of GSH in disease resistance of Arabidopsis.

Metal(loid)s and radionuclides cytotoxicity in Saccharomyces cerevisiae. Role of YCF1, glutathione and effect of buthionine sulfoximine

The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Δ ycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of γ-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of γ-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Δ ycf1.

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.

Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis.

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had >/=50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of gamma-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic gamma-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.

The homeobox gene Gsh2 is required for retinoid production in the embryonic mouse telencephalon.

We have examined the role of the homeobox gene Gsh2 in retinoid production and signaling within the ventral telencephalon of mouse embryos. Gsh2 mutants exhibit altered ventral telencephalic development, including a smaller striatum with fewer DARPP-32 neurons than wild types. We show that the expression of the retinoic acid (RA) synthesis enzyme, retinaldehyde dehydrogenase 3 (Raldh3, also known as Aldh1a3), is reduced in the lateral ganglionic eminence (LGE) of Gsh2 mutants. Moreover, using a retinoid reporter cell assay, we found that retinoid production in the Gsh2 mutants is markedly reduced. The striatal defects in Gsh2 mutants are thought to result from ectopic expression of Pax6 in the LGE. Previously, we had shown that removal of Pax6 from the Gsh2 mutant background improves the molecular identity of the LGE in these double mutants; however, Raldh3 expression is not improved. The Pax6;Gsh2 double mutants possess a larger striatum than the Gsh2 mutants, but the disproportionate reduction in DARPP-32 neurons is not improved. These findings suggest that reduced retinoid production in the Gsh2 mutant contributes to the striatal differentiation defects. As RA promotes the expression of DARPP-32 in differentiating LGE cells in vitro, we examined whether exogenous RA can improve striatal neuron differentiation in the Gsh2 mutants. Indeed, RA supplementation of Gsh2 mutants, during the period of striatal neurogenesis, results in a significant increase in DARPP-32 expression. Thus, in addition to the previously described role for Gsh2 to maintain correct molecular identity in the LGE, our results demonstrate a novel requirement of this gene for retinoid production within the ventral telencephalon.

Coupling of the Transcriptional Regulation of Glutathione Biosynthesis to the Availability of Glutathione and Methionine via the Met4 and Yap1 Transcription Factors

Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest. Here, we show that the Yap1 and Met4 transcription factors regulate the expression of gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion. Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered. Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response. The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1. In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion. Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation. These results indicate that the Yap1-dependent activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependent mechanism.

Patterning of the lateral ganglionic eminence by the Gsh1 and Gsh2 homeobox genes regulates striatal and olfactory bulb histogenesis and the growth of axons through the basal ganglia.

The function of the Gsh1 and Gsh2 homeobox transcription factors during development of the mouse telencephalon was studied using loss of function mutations. No telencephalic phenotype was observed in Gsh1 mutants, whereas Gsh2 and Gsh1/2 mutants showed progressively more severe defects in development of neurons derived from the lateral ganglionic eminence (LGE). These defects arise from abnormal dorsoventral specification of LGE progenitor cells. Mice lacking both Gsh1 and Gsh2 have severe hypoplasia of the striatum, olfactory tubercle, and interneurons that migrate from the dorsal LGE to the olfactory bulb. In addition, Gsh function is linked to the development of telencephalic dopaminergic neurons. These observations show that Gsh1 and Gsh2 have early roles in defining the identity of LGE progenitor cells. As a result of the basal ganglia defects in the Gsh1/2 mutants, there are pallial heterotopia near the cortical/subcortical limit and defects in the pathfinding of corticofugal and thalamocortical fibers. These findings highlight the developmental interdependence of adjacent telencephalic structures.

Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems.

Our studies in yeast show that there is an essential requirement for either an active thioredoxin or an active glutathione (GSH)-glutaredoxin system for cell viability. Glutathione reductase (Glr1) and thioredoxin reductase (Trr1) are key regulatory enzymes that determine the redox state of the GSH-glutaredoxin and thioredoxin systems, respectively. Here we show that Trr1 is required during normal cell growth, whereas there is no apparent requirement for Glr1. Analysis of the redox state of thioredoxins and glutaredoxins in glr1 and trr1 mutants reveals that thioredoxins are maintained independently of the glutathione system. In contrast, there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG/2GSH redox couple. We suggest that independent redox regulation of thioredoxins enables cells to survive in conditions under which the GSH-glutaredoxin system is oxidized.

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.

GSH2, a gene encoding γ-glutamylcysteine synthetase in the methylotrophic yeastHansenula polymorpha

The GSH2 gene, encoding Hansenula polymorphaγ-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121.