(A) Analysis by mathematical modeling; see Supplementary Material for a detailed model description and analysis. Simulated Fus3 activation response to pheromone progressively varying from 3 nM (lowest curve, dark blue) to 6 nM (blue), 10 nM (cyan), 100 nM (green), 1μM (yellow) and 10 μM (red). (B) Modeling analysis of pathway activation, with the concentration of active Fus3 (black) overlaid with the concentration of Sst2 (red dashed). Both curves are normalized so that their respective maxima are equal to unity. The phase shifting of Sst2 oscillations results from the interplay of pheromone induced transcription of SST2 [17] and pheromone-regulated degradation of Sst2 [22]. (C) Experimental analysis of Sst2 expression. Protein extracts were made from synchronized wild-type cells (ZH524) treated with 100 nM α-factor for the indicated times and probed with an anti-Sst2 antibody. The exposure level of this blot was chosen to maximize the dynamic range. The quantified data are shown in comparison with the time course of active Fus3 levels. (D) Modeling of the effect of Sst2 on Fus3 activation. Simulated nuclear dually-phosphorylated Fus3 plotted for different levels of Sst2 expression, with the strength decreasing from the wild type levels (blue) through intermediate levels (green) to no Sst2 (red). (E-F) Experimental (immunoblot) analysis of the effect of the removal of Fus3 negative regulators on active and total Fus3, in cells treated with 100 nM pheromone. The Fus3-GFP expression levels taken from these and replicate blots are plotted in Fig. 3C & D; (E) sst2Δ (F) msg5Δ.

(A) Immunoblot analysis of Fus3-GFP activation and expression in stimulated (100 nM) cells lacking the formin Bni1. Note that, due to weak signal in the bni1Δ strain, the time required for exposure of immunoblots to autoradiography film was about 5 to 6 times longer than similar time courses in the wild type. (B and C) Comparison of the time course of Fus3 activation (B) and total Fus3-GFP protein levels (C) in wild-type (dotted line) and bni1Δ (solid line) cells. Responses are not to scale.

(A) Experimental strategy used to monitor MAPK oscillation. Cells containing an integrated Fus3-GFP fusion protein were grown to mid-log phase, then synchronized by S-phase arrest and release. When they entered the G1 phase they were stimulated with various doses of α-factor mating pheromone for 8 hours. Samples were collected at multiple time points, and assessed by immunoblotting (to measure MAPK phosphorylation and protein abundance), microscopic examination (to measure projection formation), and other methods. (B) Representative immunoblots of wild-type (ZH524) cells treated with either 50 nM, 100 nM or 25 μM α-factor for the indicated times. After separation by SDS-PAGE in 4-20% gradient polyacrylamide gels, phospho-Fus3-GFP and phospho-Kss1 species were detected on the immunoblots with an anti-phospho-p42/44 antibody, which recognizes the phosphorylated and thereby activated isoforms of Fus3^MAPK and Kss1^MAPK [12]. The same blots were probed with an anti-GFP antibody to detect the total levels of Fus3-GFP. As loading control, blots were probed with anti-β-tubulin. (C) Quantification of Fus3 activation by scanning densitometry (ImageJ) of immunoreactive bands (anti-phospho-p42/44 antibody) on autoradiographs. The Fus3 signal was normalized to β-tubulin and expressed as % of the highest response for each pheromone concentration. Subsequently, the responses were scaled by comparing the signal intensities of the three responses for the same time points in the same autoradiography film. In this and following images, the data points were spline fitted, and each value is the average from three experiments (±SE). (D) Quantification of Fus3-GFP protein levels (anti-GFP antibody). (E) Comparison of Fus3-GFP (dotted line) and Fus1-GFP (solid line) protein levels in synchronized wild-type cells (ZH424 and ZH550 respectively) treated with 100 nM pheromone. The Fus1-GFP immunoblot is shown below. The multiple bands are caused by glycosylated isoforms [26]. In order to capture the pheromone-induced shift from the fastest to the slower migrating forms, the sum of all immunoreactive bands except the fastest migrating (i.e., lowest) Fus1-GFP band was quantified. The graph is an average of the experiment shown and two additional experiments.

Pattern of mating projection formation in cells exposed to pheromone for the indicated times overlaid with total (dashed line) levels of target gene expression (Fus3-GFP). (A-B) The effect of pheromone dose on target gene expression and projection formation in wild-type (strain ZH524) treated with (A) 100 nM pheromone, or (B) 25 μM pheromone. (C-D) The effect of the removal of Fus3 negative regulators (C) Sst2 (ZH525), or (D) Msg5 (ZH527) on targeted gene expression and projection formation in cells treated with 100 nM pheromone. Each value is the average from three experiments (±SE). The percentage of cells having 1 (red), 2 (blue), 3 (green) and 4 (magenta) projections are shown. At least one hundred cells were scored per time point for each strain. (E) Representative phase contrast images of cell morphology phenotypes of ethanol-fixed wild-type (ZH524) cells treated with 100 nM mating pheromone for 0-480 min. Bar, 10 μm. (F) Epifluorescence imaging of Fus3-GFP (green) in cells of the indicated genotype stimulated with 100 nM pheromone for 420 min.