FIGURE 5:

Localization of Bni1p to polarity sites does not require known regulatory domains. (A) The catalytic fragment (FH1–FH2; DLY13426) localizes diffusely in the nucleus and cytoplasm, but addition of N-terminal Bni1p sequences suffices to target the fragment to the bud tip and mother–bud neck, whether or not the GBD and SBD regions are included (DLY13916, 13433, 13431). Representative images of the indicated GFP-tagged constructs are shown. (B) To assess actin-independent localization, cells were treated with 200 μM Lat A at 24°C for 2.5 h (DLY13916, 13433, 13431, 13426). Bem1p-Tomato marks the polarization sites, and the indicated Bni1p constructs (except for the catalytic fragment alone) colocalize with Bem1p. Additional spots of GFP-Bni1p staining remain at old mother–bud necks, presumably reflecting problems with cytokinesis in the absence of F-actin. Scale bar, 5 μm.