FIGURE 7.
Ubiquitination of Ste4 does not regulate the magnitude and duration of pheromone-induced activation of MAP kinases. A, whole cell extracts were prepared from the wild type and the Ste4K340R mutant treated with 3 μm pheromone α factor for the indicated time, resolved by 10% SDS-PAGE, and probed with anti-phospho-p44/42 (top panel) or anti-Ste4 (bottom panel) antibodies. p-Mpk1, p-Kss1, and p-Fus3 denote phosphorylated and thus activated Mpk1, Kss1, and Fus3. B, pheromone-dependent transcriptional induction was measured following transformation of the above cells with a pheromone-responsive FUS1 promoter-GFP reporter. Cells were then treated with 3 μm α-factor for 90 min, and the resulting fluorescence in each cell was monitored by cell sorting. Pathway activation results in an increase in cells with >40 fluorescence units of activity (as indicated by an M1 bar on each graph).