Hog1 dampens Fus3 activation by targeting Ste50.

<p>Constitutive activators of mating pathway highlighted in black: (A) Ste5^CTM, a C-terminal transmembrane domain (CTM) tethers Ste5 to the plasma membrane allowing MAPK activation without receptor or G-protein. (B) Wild-type cells transformed with <i>GAL1</i>-<i>STE5^CTM</i>, <i>GAL1</i>-<i>SSK2^ΔN</i> or parent vector controls were grown in 2% galactose for 60 min followed by addition of 3 µM α factor or left untreated for 30 min. Cell lysates were resolved by 12.5% SDS-PAGE. Statistical significance was calculated using two-way ANOVA. ***, p<0.001. (C) Ste11^ΔN, constitutively active amino-terminus truncation mutant of Ste11, allowing activation without binding the upstream activator Ste20, scaffold Ste5, or adaptor Ste50. (D) Wild-type cells transformed with <i>GAL1</i>-<i>STE11^ΔN</i>, <i>GAL1</i>-<i>SSK2^ΔN</i> or vector were grown in 2% galactose for 2.5 h followed by addition of 3 µM α factor or left untreated for 30 min. Statistical significance was calculated using two-way ANOVA, ns – not significant, p>0.05. (E) Wild-type and <i>ste50^5A</i> cells grown and treated as in B. (F) <i>ste50^5A rck2</i>Δ cells grown and treated as in B. (G) Quantitative mating assay, indicated strains were mated with wild-type <i>MAT</i>α strain for 4 h on YPD or YPD+0.5 M KCl. Statistical significance was calculated using two-way ANOVA. **, p<0.01.