Regulation of Karyogamy Protein Kar4p During Mitosis and Mating in Saccharomyces cerevisiae

Abstract

The process of Saccharomyces cerevisiae mating, in which two haploid cells undergo cell wall and plasma and nuclear membrane fusion to form a diploid organism, is facilitated by a network of pheromone-induced genes. KAR4, a pheromone-induced karyogamy transcription factor, is curiously expressed in two distinct forms that appear to be functionally equivalent. However, during mitosis, Kar4p-short is very rapidly degraded, with a half-life that is one-third the magnitude of that of Kar4p-long. In addition, during mating, the short form of Kar4p not only is strongly induced, but also exhibits a stability three-fold relative to itself during mitosis. In this study, we investigate the differential turnover rates of the two forms of Kar4p, focusing on the mechanism of degradation of Kar4p-short during the mitotic state, and on the factors of the mating response that stabilize Kar4p-short. Using a transcription shut-off assay to measure turnover rates, we found that Kar4p is implicated in ubiquitin-based proteasomal degradation; the E3 ubiquitin ligase Ubr1p is specifically involved in the regulatory pathway of mitotic Kar4p-short. Furthermore, during mating, a cell-cycle independent pheromone-induced gene that is dependent upon Kar4p is responsible for stabilizing Kar4p-short. Finally, we show that phosphorylation at the S329 residue of Kar4p might be an element of the mechanism of Kar4p turnover. Through the results of this study, we have opened up several avenues to begin to understand the regulation of Kar4p in the context of the larger cell and nuclear fusion pathways, and seek to further illuminate the critical determinants of cell fate.