UBC Theses and Dissertations
Regulation of pheromone response in saccharomyces by Ste12-PRE interaction and TOR-Cdc55 signaling Su, Ting-Cheng
Ste12 is the key regulator in the yeast pheromone response pathway and works as an important model for understanding gene regulation by MAP kinase cascades. In this thesis I address how the binding strength of pheromone-response element (PRE) sequences, their orientation, and intervening nucleotide distance between two PREs govern the overall response to pheromone. I found that Ste12 binds as a monomer to a single PRE in vitro, and that two PREs upstream of a minimal core promoter cause a level of induction proportional to their relative affinity for Ste12 in vitro. Although consensus PREs are arranged in a variety of configurations in the promoters of pheromone responsive genes, I found there are severe constraints with respect to how they can be positioned in an artificial promoter to cause induction of gene expression. Two closely-spaced PREs can induce transcription in a directly-repeated or tail-to-tail orientation, while PREs separated by at least 40 nucleotides are capable of inducing transcription when oriented in a head-to-head or tail-to-tail configuration. By comparing the constraints defined by analysis of artifical promoters, I found that a single PRE can cause response to pheromone induction in combination with a properly oriented PRE-like sequence. By studying Ste12 multimerization, I found that this process might involve dephosphorylation on Ste12 to regulate the expression of pheromone-regulated genes. I discovered that Cdc55, a regulatory subunit of protein phosphatase IIA, can affect pheromone response. In the cdc55 null mutant I observed decreased expression level of a reporter gene and decreased mating efficiency. Cdc55 directly or indirectly alters the phosphorylation status of Ste12, as I observed hyperphosphorylated Ste12 in the cdc55 mutant compared to wild type. The effect of Cdc55 is independent of the pheromone response MAP kinase pathway, but was found to be controlled downstream of TOR. Analysis of artificial reporter genes and a candidate set of pheromone responsive promoters demonstrated that TOR-Cdc55 signaling regulates a distinct subset of pheromone-responsive genes. These results demonstrate a new regulatory circuit for the pheromone response controlled by the TOR signal pathway, which operates to control mating of yeast haploids in response to nutrients.
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