Figure 3.

cdc42[V36M] cells have a reduced mating efficiency and accumulate prezygotes. (A) Spot matings and growth of cdc42 mutants. Serial dilutions of mutants derived from RAY513 with indicated CDC42 or cdc42 gene (as sole copy) were spotted onto YEPD plates and incubated for 2 d. Spot matings with indicated strains are shown. Matings were carried out with the enfeebled tester JY426. (B) The expression level of the Cdc42[V36M]p is similar to that of wild type in budding and mating cells. Strains were derived from RAY1772 (MATa) and RAY513 (MATα) with indicated CDC42 or cdc42 gene (as sole copy behind its endogenous promoter on a CEN plasmid). Cell extracts of budding and mating cells were analyzed by SDS-PAGE, followed by immunoblotting and probing with anti-Cdc42p polyclonal sera. Similar results were observed in three independent experiments. (C) The cdc42[V36M] mating defect is independent of the mating type. Strains in which CDC42 or cdc42[V36M] is the sole copy behind its endogenous promoter on a CEN plasmid (derived from MATa strain RAY1772 or MATα strain RAY513) were used for quantitative matings with a wild-type tester. Values shown are the means of five or six independent experiments with SD indicated and mating efficiencies for MATa and MATα CDC42 strains (1.4 and 3.0%, respectively) set to 100%. (D) cdc42[V36M] mating mixtures accumulate prezygotes. Strains with indicated CDC42 or cdc42 as sole copy behind its endogenous promoter on a CEN plasmid derived from RAY1772 (MATa) or RAY513 (MATα) were mated with a GFP-Bud1 expressing strain (RAY1907 or RAY1487). The percentage of prezygotes formed after 4 h was determined as the number of prezygotes (fluorescence in only one cell) divided by the number of mating pairs (prezygotes and zygotes), with values representing the means of 5 to 10 determinations (n = 200–300 mating pairs) from four to eight independent matings. Error bars, SD.