Ras activity promotes MAPK focalization. (A and B) Pheromone receptor Map3-GFP (top) and MAP2K Byr1-sfGFP (bottom) in WT cell pairs before or during the fusion process (left), and at the projection tip of mutant strains (right). White arrowheads highlight focalization and red arrowheads highlight zones of broad or undetectable localization. Deletion of gap1 leads to Byr1 MAP2K focalization whether Map3 is focalized or not. (C) Percentage of cell lysis of h90 WT, rgs1Δ, and map3^dn9 mutants, with or without gap1 deletion after 14 h in MSL-N (n > 500 for three independent experiments). ***, P ≤ 2.6 × 10⁻⁵. (D) RasAct^GFP during mating in mutant strains. Note undetectable local Ras activation when the pheromone receptor is unfocalized and quasiuniform cortical activation upon gap1 deletion. (E) ^GSTRBD pulldown of protein extracts from h90 cells with indicated genotypes shifted for 4 h to mating conditions (top). Mean intensity from three independent experiments is shown (bottom). Long (20 min) and short (5 min) exposure times of pulldown samples are shown on left. *, band caused by overflow in an empty lane. Error bars, SD. Bars, 2 µm.