Figure 2.
GTP-locked Cdc42p is turned over in a Ydj1p-, Ssa1p-, Rsp5p-, and proteasome-dependent manner. (A) Level of GFP-Cdc42p (PC6454) expressed in wild-type cells (WT, S288c background, PC3288) and rsp5-1 mutant (PC3290) grown for 4 h at 30°C and shifted to 37°C for the times indicated. Values varied <25% across trials. See Fig. 1 B for details. (B) Similar experiment as A except extracts were probed with anti-Cdc42p antibodies to detect GFP-Cdc42p. Cells were grown at 37°C for 2 h. See Fig. 1 B for details. (C) GFP-Cdc42pQ61L (PC7458) levels in same cells as in A grown at the permissive temperature, 30°C. See Fig. 1 B for details. (D) Levels of ubiquitinated GFP-Cdc42pQ61L in wild-type cells (WT, S288c background, PC3288) and in the rsp5-1 mutant (PC3290) grown at 30°C for 6 h. Colored circle refers to cells expressing the indicated versions of Cdc42p and Rsp5p. See Fig. 1 A for details. (E) GFP-Cdc42pQ61L (PC7458) levels in wild-type cells (WT, S288c background, PC986) and ydj1Δ mutant (PC7657). See Fig. 1 B for details. (F) GFP-Cdc42pQ61L (PC7458) levels in wild-type cells (WT, PC6016) and ssa1Δ mutant (PC7700). See Fig. 1 B for details. (G) Wild-type cells (WT, S288c background, PC5851) and the cim3-1 mutant (PC5852) expressing GFP-Cdc42p (PC6454) were grown at 37°C and analyzed by immunoblotting at the indicated time points. See Fig. 1 B for details. (H) Fluorescence microscopy of same cells as in G grown at 30 and 37°C for 2 h. Micrographs were taken at the same exposure, the elevated signal of GFP-Cdc42p observed in the cim3-1 mutant at 37°C might be due to prolonged exposure time. Scale bar, 5 µm. (I) Comparison of relative fluorescence intensity of cells described in G. Error bars indicate SD among three biological replicates (n = 3); 35 cells were examined in each experiment. Data were analyzed by one-way ANOVA, and P values (p) were calculated using Tukey’s multiple comparison test. (J) Levels of ubiquitinated GFP-Cdc42p in wild-type cells (WT, S288c background, PC5851) and in the cim3-1 (PC5852) mutant grown at 37°C for 2 h. Colored circle refers to cells expressing the indicated versions of Cdc42p and Cim3p. See Fig. 1 A for details. (K) The pdr5Δ mutant containing GFP-Cdc42pQ61L (Q61L, PC7458) was supplemented with 0.5% ethanol (CTL, control) or 75 μM MG132 and incubated for 2 h. Values varied <25% across trials. See Fig. 1 B for details. Source data are available for this figure: SourceData F2.

GTP-locked Cdc42p is turned over in a Ydj1p-, Ssa1p-, Rsp5p-, and proteasome-dependent manner. (A) Level of GFP-Cdc42p (PC6454) expressed in wild-type cells (WT, S288c background, PC3288) and rsp5-1 mutant (PC3290) grown for 4 h at 30°C and shifted to 37°C for the times indicated. Values varied <25% across trials. See Fig. 1 B for details. (B) Similar experiment as A except extracts were probed with anti-Cdc42p antibodies to detect GFP-Cdc42p. Cells were grown at 37°C for 2 h. See Fig. 1 B for details. (C) GFP-Cdc42pQ61L (PC7458) levels in same cells as in A grown at the permissive temperature, 30°C. See Fig. 1 B for details. (D) Levels of ubiquitinated GFP-Cdc42pQ61L in wild-type cells (WT, S288c background, PC3288) and in the rsp5-1 mutant (PC3290) grown at 30°C for 6 h. Colored circle refers to cells expressing the indicated versions of Cdc42p and Rsp5p. See Fig. 1 A for details. (E) GFP-Cdc42pQ61L (PC7458) levels in wild-type cells (WT, S288c background, PC986) and ydj1Δ mutant (PC7657). See Fig. 1 B for details. (F) GFP-Cdc42pQ61L (PC7458) levels in wild-type cells (WT, PC6016) and ssa1Δ mutant (PC7700). See Fig. 1 B for details. (G) Wild-type cells (WT, S288c background, PC5851) and the cim3-1 mutant (PC5852) expressing GFP-Cdc42p (PC6454) were grown at 37°C and analyzed by immunoblotting at the indicated time points. See Fig. 1 B for details. (H) Fluorescence microscopy of same cells as in G grown at 30 and 37°C for 2 h. Micrographs were taken at the same exposure, the elevated signal of GFP-Cdc42p observed in the cim3-1 mutant at 37°C might be due to prolonged exposure time. Scale bar, 5 µm. (I) Comparison of relative fluorescence intensity of cells described in G. Error bars indicate SD among three biological replicates (n = 3); 35 cells were examined in each experiment. Data were analyzed by one-way ANOVA, and P values (p) were calculated using Tukey’s multiple comparison test. (J) Levels of ubiquitinated GFP-Cdc42p in wild-type cells (WT, S288c background, PC5851) and in the cim3-1 (PC5852) mutant grown at 37°C for 2 h. Colored circle refers to cells expressing the indicated versions of Cdc42p and Cim3p. See Fig. 1 A for details. (K) The pdr5Δ mutant containing GFP-Cdc42pQ61L (Q61L, PC7458) was supplemented with 0.5% ethanol (CTL, control) or 75 μM MG132 and incubated for 2 h. Values varied <25% across trials. See Fig. 1 B for details. Source data are available for this figure: SourceData F2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal