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Domain function and regulation of Ste12p Dambrowitz, Kirsten Amy

Abstract

Ste12p is a transcriptional activator and an effector of two MAP kinase cascades in the yeast Saccharomyces cerevisiae. Ste12p activates transcription required for both pheromone response and filamentous growth. The goal of this work was to further define the domain structure of Ste12p and the mechanisms that govern Ste12p activity; In response to mating pheromone, Ste12p activates transcription of FAR1. Farlp interacts with the cell cycle machinery in order to cause G1 growth arrest. This work showed that overexpression of Ste12p in the absence of pheromone induced G1 growth arrest that was independent of FAR1 and of transcription from pheromone responsive elements. The growth arrest did not reflect cell death and could be eliminated by overexpression of Rst1p and Rst2p, two negative regulators of Ste12p. These data indicated a novel role for Ste12p in pheromone-responsive growth arrest. Rst1p and Rst2p, two negative regulators of Ste12p, are genetically redundant and, as such, are proposed to regulate Ste12p function by a common mechanism. This study demonstrated that overexpression of amino acids 262 to 594 of Ste12p activated transcription and that concurrent overexpression of either RST1 or RST2 abrogated that activation. The region 262 to 594 was able to interact with Rst1 p, but not with Rst2p, in yeast extracts. Rst2p, in contrast, interacted with the DNA binding domain of Ste12p. Further, Rst2p, but not Rst1p, was shown to inhibit interaction of the Ste12p DNA binding domain with DNA in vitro and to inhibit the function of a Ste12pDBD-VP16 fusion in vivo. In addition, recombinant Rst1p and Rst2p were shown to interact directly with these distinct domains of Ste12p in vitro. These results showed that Rst1p and Rst2p function by non-identical mechanisms, as they interact with separate domains of Ste12p in order to inhibit its function.

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