(A) Amino acid sequence of Cdc24. The identified Cla4, Cla4 and Bem1 as well as Cdk1-Cln2 phosphosites are highlighted in green, pink and brown, respectively. PB1, PH, DH and CH domains are shown in grey, blue, yellow and orange, respectively. (B) Schematic representation of mutated phosphosites and domain organisation of cdc24-46A and cdc24-28D. The second schematic shows the phosphosites identified by Wai et al. The overlapping phosphosites identified by both studies are shown in red. Unique sites are shown in blue. (C). Indicated strains were grown at 25 or 37°C for 2 hr then Cdc24 was analysed by Western blot. Cdc24 phospho-mutant proteins were stable at 37°C in vivo. (D) The temperature-dependent growth defect of the cdc24-28D mutant cells at 37°C was partially suppressed by the addition of sorbitol to the growth media. Serial dilutions of the cells indicated were spotted onto YPD plates supplemented with 1 M sorbitol and grown at 25°C and 37°C for 2 days. (E) The indicated cells were arrested in a G1-like state with mating pheromone and released into the cell cycle synchronously. At the times shown, samples were fixed and the percentage of budded cells was plotted. At least 200 cells were counted for each strain and the experiment was conducted at 25°C.