title: Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor.
abstract: 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.
authors.affiliation: Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchetser, UK.
authors.forename: Glen L
authors.initials: GL
authors.lastname: Wheeler
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Quinn
authors.forename: Gabriel
authors.initials: G
authors.lastname: Perrone
authors.forename: Ian W
authors.initials: IW
authors.lastname: Dawes
authors.forename: Chris M
authors.initials: CM
authors.lastname: Grant
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D004151:Dipeptides; C073675:MET4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D008715:Methionine; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D008025:Ligases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; C017341:gamma-glutamylcysteine