2 NOVEL TARGETS OF THE MAP KINASE KSS1 ARE NEGATIVE REGULATORS OF INVASIVE GROWTH IN THE YEAST SACCHAROMYCES-CEREVISIAE

Haploid cells of budding yeast Saccharomyces cerevisiae respond to mat ing pheromones by inducing genes required for conjugation, arresting c ell cycle progression, and undergoing morphological changes. The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth. Both developmental altern atives to vegetative proliferation require the MAP kinase Kss1 and the transcriptional transactivator Ste12. Using a two-hybrid screen for g ene products that interact with Kss1, two homologous and previously un characterized loci (DIG1 and DIG2, for down-regulator of invasive grow th) were identified. DIG2 is pheromone-inducible, whereas DIG1 is cons titutively expressed. Dig1 colocalizes with Kss1 in the nucleus, coimm unoprecipitates with Kss1 from cell extracts in a pheromone-independen t manner, and is phosphorylated by Kss1 in immune complexes in a phero mone-stimulated manner. Kss1 binds specifically to a GST-Dig1 fusion i n the absence of any other yeast protein. Using-the two-hybrid method, both Dig1 and Dig2 also interact with the other MAP kinase of the phe romone response pathway, Fus3. However, neither dig1 or dig2 single mu tants, nor a dig1 dig2 double mutant, have a discernible effect on mat ing. In contrast, dig1 dig2 cells constitutively invade agar medium, w hereas a dig1 dig2 ste12 triple mutant does not, indicating that Dig1 and Dig2 share a role in negatively regulating the invasive growth pat hway. High-level expression of Dig1 suppresses invasive growth and als o causes cells to appear more resistant to pheromone-imposed cell cycl e arrest. Ste12 also binds specifically to GST-Dig1 in the absence of any other yeast protein. Collectively, these findings indicate that Di g1, and most likely Dig2 are physiological substrates of Kss1 and sugg est that they regulate Ste12 function by direct protein-protein intera ction.