Figure 6.

Pob1 binds to For3 and might facilitate the Cdc42-mediated relief of autoinhibition. (A) Actin staining of cdc42-879 endogenously expressing for3⁺-3GFP or for3DAD*-2GFP alleles (top). Localization of For3-3GFP or For3DAD*-2GFP in cdc42–879 cells (bottom). (B) Actin (top and middle) and calcofluor (CF) staining (bottom) of wild-type (wt), wild-type cells expressing for3DAD*, pob1-664, and pob1-664 cells expressing for3DAD* grown at 25 or 36°C. (C) Extracts from cells expressing GFP-Pob1 and For3-Myc under their endogenous promoters were immunoprecipitated using anti-GFP antibody and probed with anti-Myc antibody. Extracts were assayed for the level of GFP-Pob1 and For3-Myc by Western blot. (D) Two-hybrid analysis of the interaction between different fragments of Pob1 and For3. Several regions of the pob1⁺ ORF were cloned into pAS2 and the for3 sequence coding for the N-terminal half of For3 (amino acids 1–702), cloned into pGAD was used as bait.