Figure 1. Cdc42p is ubiquitinated, and... | Rockefeller University Press

Figure 1.

$Cdc42p is ubiquitinated, and the GTP-bound conformation is rapidly degraded compared to wild type. (A) Lysates prepared from wild-type cells (PC538) with (+) and without (−) the pGFP-Cdc42p plasmid (PC6454) were examined by co-immunoprecipitation analysis. Total protein (Total) was assessed by Ponceau S staining. Input lysates and immunoprecipitated fractions were examined by anti-GFP and anti-ubiquitin (Ub) antibodies. Co-IP Ub/GFP refers to the relative band intensity of co-immunoprecipitated Ub compared to immunoprecipitated GFP protein. Numbers indicate the mean from multiple biological replicates (n ≥ 2). Representative blots are shown. Values varied <10% across trials. IP; immunoprecipitated. (B) Levels of GFP-Cdc42p (Cdc42, PC6454) and GFP-Cdc42pQ61L (Q61L, PC7458) proteins were examined in wild-type cells (PC538). Anti-GFP and anti-Pgk1p antibodies were used. Cdc42/Pgk1 ratio refers to relative levels of GFP-Cdc42p to Pgk1p. Numbers represent the mean from multiple biological replicates (n ≥ 2). Representative blots are shown. Values varied <20% across trials. (C) Wild-type cells expressing a plasmid containing GFP-Cdc42p (Cdc42, PC6454) were incubated in SD-URA media containing 25 µg/ml of CHX and analyzed by immunoblotting at the indicated time points. Top panel, HMW products correspond to a longer exposure of the anti-GFP blot. See B for details. (D) Same as in C except cells containing the plasmid GFP-Cdc42pQ61L (Q61L, PC7458). Immunoblot analysis of C and D were taken from the same blots (same exposure) to quantify and directly compare levels of GFP-Cdc42p. See B for details. (E) Levels of GFP-Cdc42p (Cdc42, PC6454, set to 1 for time 0) and GFP-Cdc42pQ61L (Q61L, PC7458). Error bars represent the standard deviation (SD) among biological replicates (n = 2). (F) Fluorescence microscopy of wild-type cells expressing GFP-Cdc42p (Cdc42, PC6454) or GFP-Cdc42pQ61L (Q61L, PC7458). Micrographs were taken at the same exposure. Scale bar, 5 µm. (G) Comparison of relative fluorescence intensity of GFP-Cdc42p in the same cells described in F. Error bars indicate SD among three biological replicates (n = 3); 50 cells were examined in each replicate. Data were analyzed using unpaired t test (two-sided). P refers to P value. (H) Levels of ubiquitinated GFP-Cdc42p (Cdc42, PC6454) and GFP-Cdc42pQ61L (Q61L, PC7458) in wild-type cells by co-immunoprecipitation analysis. Different exposures are shown for immunoprecipitated and co-immunoprecipitated blots based on the level of the GFP-Cdc42p protein. Values varied <15% across trials. See A for details. Source data are available for this figure: SourceData F1.$

Cdc42p is ubiquitinated, and the GTP-bound conformation is rapidly degraded compared to wild type. (A) Lysates prepared from wild-type cells (PC538) with (+) and without (−) the pGFP-Cdc42p plasmid (PC6454) were examined by co-immunoprecipitation analysis. Total protein (Total) was assessed by Ponceau S staining. Input lysates and immunoprecipitated fractions were examined by anti-GFP and anti-ubiquitin (Ub) antibodies. Co-IP Ub/GFP refers to the relative band intensity of co-immunoprecipitated Ub compared to immunoprecipitated GFP protein. Numbers indicate the mean from multiple biological replicates (n ≥ 2). Representative blots are shown. Values varied <10% across trials. IP; immunoprecipitated. (B) Levels of GFP-Cdc42p (Cdc42, PC6454) and GFP-Cdc42p^Q61L (Q61L, PC7458) proteins were examined in wild-type cells (PC538). Anti-GFP and anti-Pgk1p antibodies were used. Cdc42/Pgk1 ratio refers to relative levels of GFP-Cdc42p to Pgk1p. Numbers represent the mean from multiple biological replicates (n ≥ 2). Representative blots are shown. Values varied <20% across trials. (C) Wild-type cells expressing a plasmid containing GFP-Cdc42p (Cdc42, PC6454) were incubated in SD-URA media containing 25 µg/ml of CHX and analyzed by immunoblotting at the indicated time points. Top panel, HMW products correspond to a longer exposure of the anti-GFP blot. See B for details. (D) Same as in C except cells containing the plasmid GFP-Cdc42p^Q61L (Q61L, PC7458). Immunoblot analysis of C and D were taken from the same blots (same exposure) to quantify and directly compare levels of GFP-Cdc42p. See B for details. (E) Levels of GFP-Cdc42p (Cdc42, PC6454, set to 1 for time 0) and GFP-Cdc42p^Q61L (Q61L, PC7458). Error bars represent the standard deviation (SD) among biological replicates (n = 2). (F) Fluorescence microscopy of wild-type cells expressing GFP-Cdc42p (Cdc42, PC6454) or GFP-Cdc42p^Q61L (Q61L, PC7458). Micrographs were taken at the same exposure. Scale bar, 5 µm. (G) Comparison of relative fluorescence intensity of GFP-Cdc42p in the same cells described in F. Error bars indicate SD among three biological replicates (n = 3); 50 cells were examined in each replicate. Data were analyzed using unpaired t test (two-sided). P refers to P value. (H) Levels of ubiquitinated GFP-Cdc42p (Cdc42, PC6454) and GFP-Cdc42p^Q61L (Q61L, PC7458) in wild-type cells by co-immunoprecipitation analysis. Different exposures are shown for immunoprecipitated and co-immunoprecipitated blots based on the level of the GFP-Cdc42p protein. Values varied <15% across trials. See A for details. Source data are available for this figure: SourceData F1.