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 degreesC) mutants of Saccharomyce s cerevisiae that are defective in the proper localization of the Golgi tra nsmembrane protein Emp47p, we uncovered a constitutive loss-of-function mut ation in CYS3/STR1, the gene coding for cystathionine-gamma -lyase. We show ed by immunofluorescence, sucrose-gradient analysis and quantitative Wester n analysis that the mutant mislocalized Emp47p to the vacuole at high tempe rature, while Golgi structures were apparently normal and biosynthetic rout ing of the vacuolar carboxypeptidase Y (CPY) and the plasma membrane GPI-an chored protein Gas1p were unaffected. The effect of high temperature on Emp 47p localization, as well as the temperature sensitivity of the mutant stra in on rich medium, appear to be caused by oxidative stress and are correlat ed with severe reductions in the intracellular levels of low-molecular-weig ht thiols. In accordance with this conclusion, cys3-2 mutant cells were mor e sensitive to the oxidizing agent 1-chloro-2,4-dinitrobenzene, which also aggravated the mislocalization of Emp47p observed at high temperature. Furt hermore, all the phenotypes of the mutant were completely complemented by e xogenous supply of the main low-molecular-weight thiol, glutathione (GSH) a nd, importantly, the thiol beta -mercaptoethanol reversed the temperature s ensitivity of the mutant. A comparison of our mutant with a mutant defectiv e in GSH synthesis showed that gsh1 Delta cells were similar to wild-type c ells under the stress conditions tested, with the exception of one novel ox idative stress-related phenotype that is observed in both cys3-2 and gsh1 D elta mutant cells - a defect in CDP-DAG metabolism upon shift to the nonper missive temperature. As most of the stress-related phenotypes of cys3-2 mut ant cells are more severe than those seen in gsh1 Delta cells, we conclude that cysteine as such is required and sufficient to confer some degree of p rotection from oxidative stress in yeast cells.