Heterologous localization of Cdc24 ^{Δ PB1} restores function. (A) Cdc24^38A-Snc2 and Cdc24^{37A ΔPB1}-Snc2 polarize together with Cdc42. Inverted, maximum projection images from a time lapse of cells expressing Cdc42-mCherry^SW and either Cdc24^38A or Cdc24^{37A ΔPB1} fused to GFP-Snc2 (Cdc24^38A-Snc2, DLY19558; Cdc24^{37A ΔPB1}-Snc2, DLY19560). Time in minutes, starting just before cells begin to polarize. Bar, 5 µm. (B) Cdc24^38A-Snc2 and Cdc24^{37A ΔPB1}-Snc2 are functional as the sole source of Cdc24. Wild-type and rsr1Δ cells with the indicated Cdc24 construct at the endogenous locus were spotted (10-fold serial dilutions) and incubated for 2 d at 30°C. From top: DLY17405, DLY17402, DLY19606, DLY19604, DLY18430, and DLY18603. (C) Heterologous polarization of Cdc24 bypasses the need for Rsr1 and Bem1 in polarity establishment. rsr1Δ bem1Δ cells containing the indicated Cdc24-Snc2 fusions as the only source of Cdc24 and carrying a URA3-marked BEM1 plasmid were grown to mid-log phase, spotted (10⁴, 10³, and 10² cells) onto medium with or without 5-fluoroorotic acid (to select for plasmid loss), and incubated for 2 d at 30°C. From top: DLY19826, DLY19773, and DLY19774. (D) Overexpression of Bem1 rescues the lethality of MT-Cdc24 overexpression. Cells were spotted onto plates containing the indicated concentrations of β-estradiol to induce overexpression of MT-Cdc24 alone (DLY15297) or MT-Cdc24 and Bem1 (DLY15311) and incubated for 2 d at 24°C. (E) Overexpression of Bem1 restores budding to cells overexpressing MT-Cdc24. Inverted, maximum projection images of cells incubated for 4 h in 5 nM β-estradiol medium to induce overexpression of MT-Cdc24 alone (DLY15297) or MT-Cdc24 and Bem1 (DLY15311). MT-Cdc24 itself becomes enriched in most buds of cells coexpressing Bem1. Bar, 5 µm.