Figure S4.
Cells lacking the fMAPK scaffold Bem4p show reduced levels of Cdc42p. (A) Cdc42p levels in extracts prepared from wild-type cells (WT, PC538) or mutants lacking interacting proteins (see Table S1). See Fig. S1 A for details. (B) Levels of GFP-Cdc42p in wild-type cells (WT, PC538) and the gic1Δ gic2Δ double mutant (PC7044). See Fig. S1 A for details. (C) Wild-type cells (PC986) and cells lacking Bem4p (PC4351) in the S288c background examined by IB analysis. See Fig. S1 A for details. (D) Wild-type cells (WT, PC538) and bem4Δ mutant (PC3551) expressing GFP-Cdc42p (PC6454) analyzed by IB analysis. See Fig. S1 A for details. GFP mRNA levels were examined by RT-PCR, SD < 0.2 based on two independent trials. (E) Levels of ubiquitinated GFP-Cdc42pQ61L in wild-type cells (WT) and in the bem4Δ mutant. Numbers indicate relative band intensity of co-immunoprecipitated Ub compared to immunoprecipitated GFP protein. Colored circle refers to cells expressing the indicated versions of Cdc42p and Bem4p. See Fig. 1 A for details. IP; immunoprecipitated. (F) Levels of Bem4-HA and Cdc42p in wild-type cells (WT, PC538), and in the bem4Δ mutant (PC3551) containing versions of Bem4p including Bem4p (BEM4, PC2754), Bem4p16–99 (PC4805), Bem4p99–200 (PC4807), Bem4p200–300 (PC4809), Bem4p300–400 (PC4811). See Fig. S1 A for details. (G) Fluorescence microscopy of wild-type cells (WT, PC538) and the bem4Δ mutant (PC3551) expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42pC188S (C188S, PC7350), GFP-Cdc42pD57Y (D57Y, PC7455) and Cdc42pQ61L (Q61L, PC7458). Micrographs were taken at the same exposure. Scale bar, 5 µm. (H) Levels of GFP-Cdc42p in same cells described in G grown in SD-URA media for 6 h. Top panel (HMW) corresponds to a longer exposure of the anti-GFP blot. See Fig. S1 A for details. (I) Analysis of P∼Fus3p, GFP-Cdc42p, and Pgk1p levels in wild-type cells (PC6810) expressing GFP-Cdc42p (PC6454) grown in YEPD media supplemented with 12 µM of α-factor the time indicated. Numbers indicate relative protein P∼Fus3p and GFP-Cdc42p compared to Pgk1p to time 0 and were represented in Fig. S3 I. See Fig. 3 F for details. (J) Serial dilutions of the ste4Δ mutant (PC538), ste4Δ bem4Δ (PC3551), and ste4Δ ste11Δ (PC3862) double mutants expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42pQ61L (Q61L, PC7458), or GFP-Cdc42pQ61L+TD (Q61L+TD, PC7654) were grown on SD-URA (CTL, control) and SD-URA-HIS (fMAPK reporter) media to evaluate the activity of the growth reporter FUS1-HIS3. Source data are available for this figure: SourceData FS4.

Cells lacking the fMAPK scaffold Bem4p show reduced levels of Cdc42p. (A) Cdc42p levels in extracts prepared from wild-type cells (WT, PC538) or mutants lacking interacting proteins (see Table S1). See Fig. S1 A for details. (B) Levels of GFP-Cdc42p in wild-type cells (WT, PC538) and the gic1Δ gic2Δ double mutant (PC7044). See Fig. S1 A for details. (C) Wild-type cells (PC986) and cells lacking Bem4p (PC4351) in the S288c background examined by IB analysis. See Fig. S1 A for details. (D) Wild-type cells (WT, PC538) and bem4Δ mutant (PC3551) expressing GFP-Cdc42p (PC6454) analyzed by IB analysis. See Fig. S1 A for details. GFP mRNA levels were examined by RT-PCR, SD < 0.2 based on two independent trials. (E) Levels of ubiquitinated GFP-Cdc42pQ61L in wild-type cells (WT) and in the bem4Δ mutant. Numbers indicate relative band intensity of co-immunoprecipitated Ub compared to immunoprecipitated GFP protein. Colored circle refers to cells expressing the indicated versions of Cdc42p and Bem4p. See Fig. 1 A for details. IP; immunoprecipitated. (F) Levels of Bem4-HA and Cdc42p in wild-type cells (WT, PC538), and in the bem4Δ mutant (PC3551) containing versions of Bem4p including Bem4p (BEM4, PC2754), Bem4p16–99 (PC4805), Bem4p99–200 (PC4807), Bem4p200–300 (PC4809), Bem4p300–400 (PC4811). See Fig. S1 A for details. (G) Fluorescence microscopy of wild-type cells (WT, PC538) and the bem4Δ mutant (PC3551) expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42pC188S (C188S, PC7350), GFP-Cdc42pD57Y (D57Y, PC7455) and Cdc42pQ61L (Q61L, PC7458). Micrographs were taken at the same exposure. Scale bar, 5 µm. (H) Levels of GFP-Cdc42p in same cells described in G grown in SD-URA media for 6 h. Top panel (HMW) corresponds to a longer exposure of the anti-GFP blot. See Fig. S1 A for details. (I) Analysis of P∼Fus3p, GFP-Cdc42p, and Pgk1p levels in wild-type cells (PC6810) expressing GFP-Cdc42p (PC6454) grown in YEPD media supplemented with 12 µM of α-factor the time indicated. Numbers indicate relative protein P∼Fus3p and GFP-Cdc42p compared to Pgk1p to time 0 and were represented in Fig. S3 I. See Fig. 3 F for details. (J) Serial dilutions of the ste4Δ mutant (PC538), ste4Δ bem4Δ (PC3551), and ste4Δ ste11Δ (PC3862) double mutants expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42pQ61L (Q61L, PC7458), or GFP-Cdc42pQ61L+TD (Q61L+TD, PC7654) were grown on SD-URA (CTL, control) and SD-URA-HIS (fMAPK reporter) media to evaluate the activity of the growth reporter FUS1-HIS3. Source data are available for this figure: SourceData FS4.

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