Figure 3.
Lysine residues are required for GTP-locked Cdc42p turnover. Turnover of Cdc42p differentially impacts MAPK pathway signaling. (A) Two Cdc42p-depenedent MAP kinase pathways. fMAPK- (red) and mating- (green) specific components. Grey, common components. (B) Left: Microscopic examination of wild-type cells (WT, PC538), and the ydj1Δ (PC7616), ssa1Δ (PC7621), tec1Δ (PC6102), tec1Δ ydj1Δ (PC7820), and the tec1Δ ssa1Δ (PC7819) double mutants grown on YEPD plates for 16 h. Right: Elongated cells expressed as a percentage for the same strains. A cell was considered elongated with >1.40 of length/width ratio. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01, all samples were compared to WT) was calculated using Tukey’s multiple comparison test. (C) Top left: Halo formation in response to α-factor for wild-type cells (WT, PC6810), and the ydj1Δ (PC7619), ssa1Δ (PC7623), and ste11Δ (PC6604) mutants. Cells were grown for 16 h, spread on YEPD media, and α-factor was spotted at two concentrations on the surface, 2 and 6 µM, to study cell-cycle arrest. Bottom: Phenotypes of same strains grown for 3 h in SD-URA media supplemented with 6 µM of α-factor. Shmooing cells were counted and represented as a percentage on the graph at right. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01, all samples were compared to WT) was calculated using Tukey’s multiple comparison test. (D) Wild-type cells (PC538) expressing GFP-Cdc42pQ61L (No change, PC7458), GFP-Cdc42pQ61L,K94R,K96R (PC7662), GFP-Cdc42pQ61L,K150R,K153R (PC7664), GFP-Cdc42pQ61L,K183R,K184R,K186R,K187R (PC7665), pGFP-Cdc42pK5R,Q61L,K150R,K153R,K166R (PC7667), pGFP-Cdc42pK5R,Q61L,K94R,K96R,K123R,K128R,K150R,K153R,K166R,K183R,K184R,K186R,K187R (12KR, PC7666), pGFP-Cdc42pK5R,Q61L,K123R,K128R,K166R (PC7651), and pGFP-Cdc42pK5R,Q61L,K94R,K96R (Q61L+TD, PC7654) were grown in SD-URA for 6 h and analyzed by immunoblotting. Values varied <25% across trials. See Fig. 1 B for details. (E) Predicted structure of Cdc42p. Blue, lysines required for GTP-Cdc42p turnover; magenta, effector-binding domain or P-loop. (F) Wild-type cells (PC6810) expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42pQ61L (Q61L, PC7458) or GFP-Cdc42pQ61L+TD (Q61L+TD, PC7654) were grown for 6 h in SD-URA media. Numbers indicate relative P∼Kss1p, P∼Fus3p and GFP-Cdc42p levels compared to Pgk1p using Q61L as the control condition. Anti-phospho-p44/42, anti-GFP, and anti-Pgk1 antibodies were used. Representative blots are shown. Values varied <25% across trials. (G) Left: Wild-type cells (WT, PC538) and the tec1Δ mutant (PC6102) expressing the plasmids described in F were grown to mid-log phase and examined by microscopy. Cells that represent the most common phenotype are shown in the figure. Scale bar, 5 µm. Right: Elongated cells expressed as a percentage for the same strains shown on the left. A cell was considered elongated with >1.40 of length/width ratio. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01) was calculated using Tukey’s multiple comparison test. n.s., not significant. (H) Top left: Halo formation in response to α-factor of wild-type cells (WT, PC6810) and the ste11Δ mutant (PC6604) expressing the same plasmids as in F. Cells grown for 16 h in the selective SD-URA media were spread on SD-URA media, and α-factor was spotted at two concentrations on the surface, 2 and 6 µM, to study cell-cycle arrest. Bottom: Phenotypes of same cells expressing plasmids described in F grown for 2 h in SD-URA media supplemented with 6 µM of α-factor, images represent the most common phenotype. Shmooing cells were counted and represented as a percentage on the graph at right. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01) was calculated using Tukey’s multiple comparison test. Comparisons were made to WT cells expressing Cdc42 unless otherwise indicated. Source data are available for this figure: SourceData F3.

Lysine residues are required for GTP-locked Cdc42p turnover. Turnover of Cdc42p differentially impacts MAPK pathway signaling. (A) Two Cdc42p-depenedent MAP kinase pathways. fMAPK- (red) and mating- (green) specific components. Grey, common components. (B) Left: Microscopic examination of wild-type cells (WT, PC538), and the ydj1Δ (PC7616), ssa1Δ (PC7621), tec1Δ (PC6102), tec1Δ ydj1Δ (PC7820), and the tec1Δ ssa1Δ (PC7819) double mutants grown on YEPD plates for 16 h. Right: Elongated cells expressed as a percentage for the same strains. A cell was considered elongated with >1.40 of length/width ratio. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01, all samples were compared to WT) was calculated using Tukey’s multiple comparison test. (C) Top left: Halo formation in response to α-factor for wild-type cells (WT, PC6810), and the ydj1Δ (PC7619), ssa1Δ (PC7623), and ste11Δ (PC6604) mutants. Cells were grown for 16 h, spread on YEPD media, and α-factor was spotted at two concentrations on the surface, 2 and 6 µM, to study cell-cycle arrest. Bottom: Phenotypes of same strains grown for 3 h in SD-URA media supplemented with 6 µM of α-factor. Shmooing cells were counted and represented as a percentage on the graph at right. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01, all samples were compared to WT) was calculated using Tukey’s multiple comparison test. (D) Wild-type cells (PC538) expressing GFP-Cdc42pQ61L (No change, PC7458), GFP-Cdc42pQ61L,K94R,K96R (PC7662), GFP-Cdc42pQ61L,K150R,K153R (PC7664), GFP-Cdc42pQ61L,K183R,K184R,K186R,K187R (PC7665), pGFP-Cdc42pK5R,Q61L,K150R,K153R,K166R (PC7667), pGFP-Cdc42pK5R,Q61L,K94R,K96R,K123R,K128R,K150R,K153R,K166R,K183R,K184R,K186R,K187R (12KR, PC7666), pGFP-Cdc42pK5R,Q61L,K123R,K128R,K166R (PC7651), and pGFP-Cdc42pK5R,Q61L,K94R,K96R (Q61L+TD, PC7654) were grown in SD-URA for 6 h and analyzed by immunoblotting. Values varied <25% across trials. See Fig. 1 B for details. (E) Predicted structure of Cdc42p. Blue, lysines required for GTP-Cdc42p turnover; magenta, effector-binding domain or P-loop. (F) Wild-type cells (PC6810) expressing GFP-Cdc42p (Cdc42, PC6454), GFP-Cdc42pQ61L (Q61L, PC7458) or GFP-Cdc42pQ61L+TD (Q61L+TD, PC7654) were grown for 6 h in SD-URA media. Numbers indicate relative P∼Kss1p, P∼Fus3p and GFP-Cdc42p levels compared to Pgk1p using Q61L as the control condition. Anti-phospho-p44/42, anti-GFP, and anti-Pgk1 antibodies were used. Representative blots are shown. Values varied <25% across trials. (G) Left: Wild-type cells (WT, PC538) and the tec1Δ mutant (PC6102) expressing the plasmids described in F were grown to mid-log phase and examined by microscopy. Cells that represent the most common phenotype are shown in the figure. Scale bar, 5 µm. Right: Elongated cells expressed as a percentage for the same strains shown on the left. A cell was considered elongated with >1.40 of length/width ratio. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01) was calculated using Tukey’s multiple comparison test. n.s., not significant. (H) Top left: Halo formation in response to α-factor of wild-type cells (WT, PC6810) and the ste11Δ mutant (PC6604) expressing the same plasmids as in F. Cells grown for 16 h in the selective SD-URA media were spread on SD-URA media, and α-factor was spotted at two concentrations on the surface, 2 and 6 µM, to study cell-cycle arrest. Bottom: Phenotypes of same cells expressing plasmids described in F grown for 2 h in SD-URA media supplemented with 6 µM of α-factor, images represent the most common phenotype. Shmooing cells were counted and represented as a percentage on the graph at right. Error bars represent the SD from three biological replicates (n = 3); 200 cells were analyzed in each replicate. Data were analyzed by one-way ANOVA, and the P value (asterisk < 0.01) was calculated using Tukey’s multiple comparison test. Comparisons were made to WT cells expressing Cdc42 unless otherwise indicated. Source data are available for this figure: SourceData F3.

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