FIGURE 3:

Negative feedback regulation of Cdc24 depends on a functional Bem1-scaffolded signaling module. (A) The protein level of Cdc24-3xHA expressed under control of its endogenous promoter was determined in cells that overexpress constitutive active Rac1^Q61L, dominant negative Rac1^T17N, and constitutive active Cla4ΔCRIB kinase. Cells were grown in noninducing medium, and proteins were isolated before induction (OFF) and 8 h after induction (ON). (B) The protein level of constitutively expressed mutant Cdc24^T285A-GFP was determined in comparison with the level of wild-type Cdc24-GFP expressed under the same conditions. (C) The effect of Rac1 overexpression on the protein level of Cdc24-GFP was determined in the presence (+) or absence (−) of either the Rac1 effector Cla4 or the scaffolding protein Bem1. Rac1 was ectopically expressed under control of the nitrate-inducible nar1-promoter in wild type, in Δcla4 mutants, and in a conditional bem1^crg mutant strain that expresses the bem1 gene under control of the arabinose-inducible crg promoter. Cells were grown in liquid minimal medium containing glucose (−) or arabinose (+) as carbon source and ammonium (OFF) or nitrate (ON) as nitrogen source. (D) Rac1-GTP needs both the scaffold Bem1 and the effector Cla4 to target Cdc24 for degradation. (E) The protein level of Cdc24-GFP was determined before (OFF) and after induction of an ectopic copy of constitutive active Cla4ΔCRIB kinase in the wild type, in the rac1 deletion mutant, and in the conditional bem1 mutant. Growth conditions were the same as described in C. (F) Constitutively active Cla4ΔCRIB needs the bridging function of Bem1 to induce degradation of Cdc24. (G) Phenotypes of AB31, AB31Δcla4, and AB31cla4-AS before (0 h) and after induction of bE1/bW2 overexpression (8 h). Strain AB31cla4-AS expresses the analogue-sensitive mutant cla4^M629A, which can be inhibited by 1NM-PP1. Scale bars, 10 μm. (H) Determination of endogenous Cdc24-3x-HA levels in the cells shown in G.