The N-terminal 100 residues of Bem3p interact with the C-terminal coiled-coil regions of Epo1p. (A) Split-Ub assay as in Fig. 1 B but with cells coexpressing Epo1CRU and Nub fusions to Bem3p and its fragments. The Nub fusion to Guk1p should not interact and served as a control for the specificities of the observed interactions. (B) Cartoon indicating the positions of the Nub fragments and the domains of Bem3p. GAP, GTPase-activating domain. (C) As in Fig. 1 F, but with protein extracts of bacterial cells expressing his6-Epo1852–943 (lanes 1, 3, and 4) or his6-Epo1761–867 (lanes 2, 5, and 6) that were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 3 and 5) or GST-Bem31–100 (lanes 4 and 6). The vertical line indicates the removal of a lane loaded with a molecular mass marker. The inputs of GST and GST-Bem31–100 are shown as lanes 7 and 8 of a Coomassie-stained gel. Arrows indicate the running positions of GST-Bem31–100 and GST.
