Figure 1.

Interaction between Bni4 and Glc7. (A) Two-hybrid analysis of Bni4 and Glc7. Strain PJ69-4A was cotransformed with pHH149, encoding Glc7 fused to the Gal4 DNA-binding domain, and either pAR5 (BNI4 aa 70–892), pAR14 (BNI4 aa 1–779), pAR16 (BNI4 aa 1–892), or pAR19 (bni4^V-A/F-A aa 70–892), which encode the indicated Bni4 variants. Two representative transformants were plated on synthetic medium lacking tryptophane and leucine (SC-Trp/Leu) to select for the two plasmids. The two-hybrid interaction was assayed by replica plating the transformants onto SC-Ade, SC-His, or SC-His supplemented with 10 mM 3-amino-triazole (−His + 3AT). (B) Coimmunoprecipitation of Bni4 and Glc7. Strain KT1927 (GFP-GLC7, bni4::TRP1) was transformed with p366 (BNI4), the empty vector pRS316, and pAR17 (bni4^V-A/F-A). Immunoprecipitates were subjected to PAGE and immunoblots were probed using anti-GFP antibody (αGFP) or anti-Bni4 antibody (αBni4). (C) Strains YLK29 (Bni4-CFP, YFP-Glc7), YLK27 (Bni4-CFP) and YAB122 (YFP-Glc7) were imaged with CFP, YFP, and FRET filter sets. Bar, 5 μm.