The Regulation of Transcription Factor Kar4p in the Mating and Mitotic Growth of Saccharomyces cerevisiae

Abstract

Mating of two haploid Saccharomyces cerevisiae cells entails plasma membrane fusion followed by karyogamy (fusion of two haploid nuclei). The process requires induction of a multitude of mating-specific elements, including the transcription factor Kar4p. KAR4 is expressed in two functionally equivalent forms: a constitutively expressed long form that predominates during vegetative growth, and a slightly shorter form that is highly induced by mating pheromone. During mitosis, Kar4p-short is turned over a rate three times that of its rate of turnover in mating. This study investigates the differential turnover rates of the two forms of Kar4p, focusing both on the elements that are necessary for the mitotic instability of Kar4p-short, as well as elucidating those that are necessary for its pheromone-induced stabilization. Motivated by recent work suggesting that the E3 ubiquitin ligase Ubr1p is involved in the degradation of Kar4p- short through ubiquitin-proteasome proteolysis, I confirm the necessity of the UBR1 for a high turnover rate in the mitotic state, using a transcription shut-off assay to measure turnover rates. Additionally, I show that the Ubr1p enzyme targets Kar4p-short for ubiquitination through the N-end rule and that the stabilization of the short form is likely due to the pheromone response causing the downregulation of Ubr1p activity. Finally, after confirming that the mating-induced stabilization of Kar4p-short depends on a mating factor downstream of Kar4p, the relatively uncharacterized SRL4 gene is identified as being necessary for such stabilization. Although further questions remain to be answered, these results paint a more complete picture on how such a complicated method of differential regulation of Kar4p is achieved.