Requirement for BEM1 and RSR1 and biochemical assay of the effect of Bem1 on Cdc24 GEF activity. (A) Tetrads from rsr1Δ/RSR1 bem1Δ/BEM1 S288C diploids (DLY17480). rsr1Δ bem1Δ spores often fail to produce viable colonies at 24°C. Green, viable rsr1Δ bem1Δ spores; red, inviable rsr1Δ bem1Δ spores. At 37°C, both RSR1 bem1Δ and rsr1Δ bem1Δ spores are inviable. (B) DIC images of rsr1Δ BEM1 and viable rsr1Δ bem1Δ cells (DLY18495 and DLY18459) expressing the histone H2B tagged with mCherry. These cells were generated from a transformed diploid strain constructed by using haploids isolated from the tetrads in A. Scale bar, 5 µm. (C and D) Cdc24 GEF activity in vitro. Arbitrary units (AU): amount of radioactive GTP loaded on Cdc42 divided by the amount of HA-Cdc24 in the immunoprecipitate (inset). Activity is normalized to the wild-type Cdc24. (C) Cdc42 GTP loading by Cdc24 isolated from yeast: wild-type, DLY15284; Cdc24^KR (no Bem1 binding), DLY17327; Cdc24 from bem1Δ cells, DLY15299. (D) Wild-type, DLY15819; Cdc24^ΔPB1 (lacks putative autoinhibition), DLY15818; Cdc24^AA (catalytically dead control), DLY15817. Mean ± SEM (n = 3).