We have not ruled out that the Mepiroxol altered pathway output we observed is due to structural changes unrelated to phosphorylation and/or altered protein/protein interactions caused by the alanine substitutions. In strains expressing a protein with phosphorylation site point mutations, no cell will have the mutated site phosphorylated. In the reference strain in any given single cell, the fraction of the population of molecules of the protein with the site phosphorylated is unknown. We can thus expect that the effect of any phosphorylation site on signaling pathway output will be incompletely penetrant. The quantitative phenotypes we measured in the mutant strains �C measurements made comparing the population of mutant cells to the heterogenous reference cells �C may therefore underestimate the effect that these phosphorylation sites exert on the activity of the proteins they modify. On Ste12, we identified 2 putative phosphorylation sites, S400 and T525, that each contribute,25% to the transcriptional activity of Ste12 across a dose response of pheromone. Based on Tulathromycin B neighboring sequence context, these sites are likely to be MAPK targets and we demonstrated that they both contribute to the appropriate transcriptional output to a given dose of pheromone. The conservation of these sites among closely related yeast species and the quantitative agreement of the computational model with the experimental results lend credence to this notion. While the 25% gain in activity afforded by these sites is unlikely to be absolutely required for mating in laboratory settings, such a quantitative increase in output may have conferred competitive fitness over evolutionary timescales. The adaptor protein Ste50 links the 
MAP3K Ste11 to active Cdc42 and Ste20, thereby localizing Ste11 to its upstream activators at the plasma membrane. Ste11 then signals through different MAPK cascades via its association with different scaffold proteins: Ste5 directs signaling to the pheromone pathway, while Pbs2 directs signaling to the hyper-osmotic stress pathway. Several groups have suggested that phosphorylation of Ste50, possibly on S202, may help determine how much signal from active Ste11 goes to the pheromone pathway, and how much goes to the hyperosmotic stress pathway. Mechanistically, phosphorylation of Ste50 on S202 may influence pathway choice by regulating protein-protein interactions with membrane anchoring factors that associate with the pathway-specific scaffolds. Here we propose that phosphorylation of S202 on Ste50 anchors a negative feedback loop that inhibits pheromone pathway output in response to low doses of pheromone. Thus, phosphorylation of S202 may serve to dampen the threshold required to activate a full-fledged pheromone response. This negative feedback loop could be relevant when mating partners are scarce and commitment to the mating program would waste resources, and also when both pheromone and high osmolarity are present to ensure that enough signaling bandwidth is available to trigger the response to osmotic stress. For the redundant repressor protein Dig1, we utilized a sensitized genetic background in which CFP-tagged Ste12 requires Dig1 for its full activity to increase the likelihood of revealing functional roles for Dig1 mutants without having to delete the paralogous repressor Dig2.