THE RESPONSE REGULATOR-LIKE PROTEIN POS9 SKN7 OF SACCHAROMYCES-CEREVISIAE IS INVOLVED IN OXIDATIVE STRESS RESISTANCE/

We have isolated mutants of Saccharomyces cerevisiae with an increased sensitivity to oxidative stress. All pos9 mutants (pos for peroxide s ensitivity) were hypersensitive to methylviologene, hyperbaric oxygen or hydrogen peroxide, but grew similarly to the wild-type under all ot her conditions tested. Isolation and sequencing of the respective POS9 gene revealed that it was identical to SKN7. The predicted Skn7/Pos9 protein possesses a domain with high homology to prokaryotic response regulators. These regulatory proteins are part of a simple signalling cascade termed a ''two-component system'', where a phosphorylation sig nal of a histidine kinase is transferred to a conserved aspartate resi due of the response regulator. To test the functional role of the resp ective aspartate residue of Skn7/Pos9 protein in oxidative stress, we mutagenized this residue in vitro to alanine, arginine and glutamate. Only the glutamate allele (D-427 to E) was able to rescue the hydrogen peroxide-sensitivity of pos9 mutants. By fusion experiments with the Gal4 DNA-binding domain we identified the isolated response regulator- like domain as a novel eukaryotic domain sufficient for gene activatio n. Whereas this hybrid protein activated transcription of a lacZ repor ter gene under aerobic conditions, no activation was observed under an aerobic conditions, indicating that the response regulator domain is i nvolved in a signalling reaction. Two-hybrid investigations also sugge st an oligomerization of the Pos9 protein. Our results indicate that a two-component system is involved in the oxidative-stress response of yeast.