title: 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.
abstract: 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.
authors.affiliation: Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, 37070 Göttingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Matiach
authors.forename: S
authors.initials: S
authors.lastname: Schröder-Köhne
mesh_terms: D002455:Cell Division; D003542:Cystathionine gamma-Lyase; D003545:Cysteine / Q000494:pharmacology; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D050356:Lipid Metabolism; D008565:Membrane Proteins; D016296:Mutagenesis; D009154:Mutation; D018384:Oxidative Stress; D010641:Phenotype; D021381:Protein Transport; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins
chemical_list: D003567:Cytidine Diphosphate Diglycerides; D004268:DNA-Binding Proteins; C096914:EMP47 protein, S cerevisiae; D005656:Fungal Proteins; D018398:Homeodomain Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D005020:Ethyl Methanesulfonate; C073674:CYS3 protein, S cerevisiae; D003542:Cystathionine gamma-Lyase; C490862:GSH1 protein, S cerevisiae; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine