Cdc24 localization is necessary for Cdc24 function. (A) Cdc24^ΔPB1 is unable to function as the sole source of Cdc24. Left: Tetrads of heterozygous diploids expressing Cdc24^ΔPB1-3HA (DLY15184) or Cdc24-3HA (DLY15073) at the CDC24 locus were grown at 24°C for 2 d. (right) Western blot of Cdc24 from the heterozygous diploids. (B) Western blot comparing expression of nonphosphorylatable, membrane-targeted MT-Cdc24^38A (DLY18203) and MT-Cdc24^{37A ΔPB1} (DLY18490) relative to wild-type Cdc24 (DLY12338). (C) MT-Cdc24 localizes to the plasma membrane. Inverted single-slice-scanning confocal image of heterozygous diploid cells expressing MT-Cdc24^38A (DLY18203) or MT-Cdc24^{37A ΔPB1} (DLY18490). Bar, 5 µm. (D) MT Cdc24^{37A ΔPB1} is unable to function as the sole source of Cdc24. Tetrads of heterozygous cdc24Δ/CDC24 diploids expressing MT-Cdc24^38A (DLY18203) or MT-Cdc24^{37A ΔPB1} (DLY18490) were grown at 24°C for 3 d. Green boxes indicate viable cdc24Δ colonies, showing rescue by MT-Cdc24^38A. (E) MT Cdc24^{37A ΔPB1} causes arrest as large, round, unbudded cells. DIC images of cdc24-1 cells expressing MT-Cdc24^38A (DLY19120) or MT-Cdc24^{37A ΔPB1} (DLY19218) shifted to 37°C for 6 h. Bar, 5 µm. (F) MT Cdc24^{37A ΔPB1} is unable to rescue cdc24-1. Cells were spotted onto plates (10-fold serial dilutions) and incubated for 3 d. From top: DLY8502, DLY19122, DLY19498, DLY8534, DLY19120, and DLY19218.