Oxidative Stress ActivatesFUS1andRLM1Transcription in the YeastSaccharomyces cerevisiaein an Oxidant-dependent Manner

Abstract

Mating in haploid Saccharomyces cerevisiae occurs after activation of the pheromone response pathway. Biochemical components of this pathway are involved in other yeast signal transduction networks. To understand more about the coordination between signaling pathways, we used a "chemical genetic" approach, searching for compounds that would activate the pheromone-responsive gene FUS1 and RLM1, a reporter for the cell integrity pathway. We found that catecholamines (L-3,4-hydroxyphenylalanine [L-dopa], dopamine, adrenaline, and noradrenaline) elevate FUS1 and RLM1 transcription. N-Acetyl-cysteine, a powerful antioxidant in yeast, completely reversed this effect, suggesting that FUS1 and RLM1 activation in response to catecholamines is a result of oxidative stress. The oxidant hydrogen peroxide also was found to activate transcription of an RLM1 reporter. Further genetic analysis combined with immunoblotting revealed that Kss1, one of the mating mitogen-activated protein kinases (MAPKs), and Mpk1, an MAPK of the cell integrity pathway, participated in L-dopa-induced stimulation of FUS1 and RLM1 transcription. We also report that Mpk1 and Hog1, the high osmolarity MAPK, were phosphorylated upon induction by hydrogen peroxide. Together, our results demonstrate that cells respond to oxidative stress via different signal transduction machinery dependent upon the nature of the oxidant. INTRODUCTIONIn the haploid cells of the yeast Saccharomyces cerevisiae, four essential MAPK cascades (Figure 1) respond to different external signals to mediate specific responses. The mating pathway is activated by peptide pheromones and induces cell-cycle arrest and the morphological changes required for mating (Elion, 1998;Gustin et al., 1998). The invasive growth pathway is activated by starvation and induces foraging into the agar substratum. The high osmolarity glycerol (HOG) pathway increases intracellular glycerol levels in response to hypertonic stress, whereas the cell integrity pathway is activated by hypotonic stress, heat shock, or impaired cell wall synthesis. Recently, an additional MAPK cascade has been described: the Kss1 vegetative growth pathway (Lee and Elion, 1999). The Kss1 pathway may be activated by cell wall stress or changes in osmolarity (Cullen et al., 2000).The mating pathway is the most studied cellular response to an external signal. As a relatively simple G protein-coupled cascade, it is a widely used model to study mammalian G protein-coupled receptors. The mating cascade includes pheromone receptors (Ste2 and Ste3), G proteins (Gpa1, Ste4, and Ste18), the p21 activating protein kinase Ste20, a mitogen-activated protein kinase kinase kinase (MAPKKK) Ste11, a mitogen-activated protein kinase kinase (MAPKK) Ste7, a scaffolding protein Ste5 and two MAPKs (Fus3 and Kss1) (Gustin et al., 1998;Madhani and Fink, 1998;Farley et al., 1999). Targets of the terminal MAPK include Ste12, a factor required for transcription of pheromone-responsive genes, and Far1, a bifunctional protein required for polarization and G1...

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