Oxidative stress responses and sumoylation in Saccharomyces cerevisiae

This thesis is concerned with cellular responses to stress including the adaptive response to H2O2, and the cellular roles of sumoylation in stress responses. 286 H2O2-sensitive Saccharomyces cerevisiae deletion mutants were screened and YAP1, SKN7, GAL11, RPE1, TKL1, IDP1 were identified to be important for adaptation to H2O2. The mutants fell into two groups based on their responses to acute and chronic doses of H2O2. Transcription factors Yap1p, Skn7p and Gal11p were important for both acute and chronic responses to H2O2. Yap1p and Skn7p were needed for up-regulation of anti-oxidant functions rather than generation of NADPH or glutathione. Adaptation was reduced in strains deleted for GPX3 and YBP1, which are involved in sensing H2O2 and activating Yap1p, but to a lesser extent than YAP1 deletion. RPE1, TKL1 and IDP1 deletants affected in NADPH production were chronically sensitive to H2O2, but resistant to an acute dose and other mutants affected in NADPH generation were also affected in adaptation. These mutants overproduced reduced glutathione (GSH) but maintained normal cellular redox homeostasis. Over-production of GSH was not regulated by transcription of the gene encoding -glutamylcysteine-synthetase. The Skn7p transcription factor is therefore important for the adaptive response to oxidative stress-induced by H2O2, and NADPH generation is also required for adaptation.The roles of sumoylation in stress responses and transcriptional regulation were examined by deleting the SUMO ligases Siz1p and Siz2p. Siz1p is required for tolerance to copper ions and DNA damage repair. Siz2p is involved in repression of stress responses, particularly oxidative stress and is required for activation of nucleotide and RNA metabolism, DNA processing and cell division. Both Siz1p and Siz2p act in parallel in the repressing heat-shock responses and in reducing chronological life span. Genome-wide transcriptional analysis showed that Siz1p and Siz2p repress the mitochondrial retrograde pathway and arginine biosynthesis, while activating some carbon and nitrogen metabolism genes. Sumoylation of proteins in the wild type was induced by nitrogen starvation or mitochondrial inhibition during the initial treatment. However, nitrogen starvation led to some protein degradation, while the SUMO-conjugated proteins were recycled in cells with disrupted mitochondrial functions