Phosphorylation of the RING-H2 domains of Ste5 and Far1 inhibits signaling and oriented cell polarity. (A) Extracts prepared from ste5Δ cells expressing 3xGFP-tagged WT Ste5, nonphosphorylatable Ste5^S185A, or phospho-mimicking Ste5^S185D or harboring an empty control plasmid were analyzed by immunoblotting using anti-GFP antibodies. Pgk1 controls equal loading. (B) Halo assays were used to assess cell cycle arrest in response to α-factor for ste5Δ cells expressing Ste5 and control constructs as in A. (C) Pheromone-dependent gene expression of ste5Δ cells harboring a FIG1-qV reporter and the indicated WT Ste5 (diamond), Ste5^S185A (square), or Ste5^S185D (triangle) was determined by FACS analysis and plotted in arbitrary units at the times indicated (minutes) after α-factor addition. (D) Microfluidic chambers generated a 0–80-nM α-factor gradient. The angle of the polarity site with respect to the α-factor gradient (schema) was measured for far1Δ strains expressing WT Far1, nonphosphorylatable Far1^3A, or phospho-mimicking Far1^3E. Results are expressed as percentages and binned into 45° incremental distances (Hegemann et al., 2015).