Two phosphorylation sites in Ste12 are required for full transcriptional induction.

<p>A. Alignment of 2 tryptic phospho-peptides across 6 yeast species. Possible phosphorylation sites (serines and threonines) and minimal MAPK consensus sequences (S/T,P) are in bold. B. Western blots of lysate from yeast strains containing the indicated wild type or mutant Ste12, treated or not treated with 1 µM alpha factor for 15 minutes probed with anti-Ste12 and anti-GAPDH. 4x mut is Ste12^{S400A,S402A,T405A,S406A}. C. Histograms of the single cell (n > 2000 cells) pheromone response measured by p<i>PRM1</i>-mCherry fluorescence. Cells were treated with 4 nM alpha factor for 3 hrs followed by cycloheximide for 2 hrs, and measured by flow cytometry. D. Dose responses of cells treated with 12 concentrations of pheromone as described in C. Figure plots means of the unimodal distributions. Error bars depict the standard error of the mean. E. Cartoon model of the function of the phosphorylation of S400 and T525 F. Simulation of the ODE model across a dose response of pheromone.