Bem1 localization is necessary for Bem1 function. (A) Wild-type Bem1 and a Cdc42 interaction–defective mutant (Bem1^N253D) polarize with similar dynamics. Top: Inverted maximum projection images at 45-s intervals comparing Bem1-GFP and Bem1^N253D-GFP homozygous diploids (DLY17251 and DLY19400). Time point starting just before polarization. Middle: Quantification of Bem1-GFP or Bem1^N253D-GFP cluster intensity in the cells above. Bottom: Additional quantifications of different cells. (B) Anchor-away technique: FRB (orange diamond) and GFP are fused to the C terminus of Bem1, and the FKBP12 domain (blue arrow) is fused to the C terminus of the large ribosomal subunit Rpl13a. In the absence of rapamycin, Bem1 localizes normally to the polarity site (left). In the presence of rapamycin, FKBP12 and FRB interact, sequestering Bem1 to the ribosomes (right). PM, plasma membrane. (C) Sequestering Bem1 to ribosomes blocks Bem1 polarization. Upon addition of rapamycin, Bem1-FRB-GFP is sequestered to the cytoplasm. Inverted, maximum projection images of cells (DLY15549) expressing Bem1-FRB in DMSO or 50 µg/ml rapamycin. (D) Sequestering Bem1 to the cytoplasm blocks budding. DIC images of rsr1Δ cells expressing Bem1-FRB-GFP (DLY15971) before and after 6-h incubation on a 50-µg/ml rapamycin slab. See Video 1. (E) Bem1 fused to a plasma membrane protein (Snc2^V39A,M42A) localizes to the plasma membrane. Inverted single-slice-scanning confocal image of diploid cells expressing Bem1-GFP-FRB and Bem1-GFP-Snc2^V39A,M42A (DLY18783). (F) Localization of Bem1 to the plasma membrane does not rescue budding. DIC images of cells expressing Bem1-FRB-GFP and Bem1-GFP-Snc2^V39A,M42A (DLY19244) before and after 12-h incubation on a 50-µg/ml rapamycin slab. See Video 2. Bars, 5 µm.