A mutation in the CBD of Myo2 that impairs the interaction with Pea2. (A) Summary of the mutation analysis. Left: Structure of the surface of CBD (Pashkova et al., 2006). Residues that exclusively contact Sec4 are colored yellow; those that exclusively contact Pea2 are colored red. Residues contacting both proteins are orange. Residues with no impact on binding are blue. Right: Blow-up of the Myo2 interface showing position and identities of exchanged residues. Color code as in left panel. (B) Dissection of Pea2 binding site. Yeast cells containing centromeric CBDCRU plasmids with the indicated point mutations and expressing N_ub-Pea2 or N_ub-Sec4 were analyzed as in Fig. 1 C. (C) Split-Ub assay as in B but with cells expressing N_ub-Sec4 or N_ub-Pea2 and CRU fusions to the genomic MYO2 carrying no exchange (upper panel), the R1419A exchange (middle panel), or the R1419D exchange (lower panel). (D) Myo2_RD affects Pea2 binding in vitro. Upper panel: 6xHis fusion to CBD (lanes 1, 3, and 5) or CBD_RD (lanes 2, 4, and 6) were purified from E. coli and incubated with GST-Pea2_221–350 (lanes 3 and 4) or GST-coupled beads (lanes 5 and 6). Glutathione eluates were separated by SDS-PAGE, transferred onto nitrocellulose, and stained with anti-His antibody. Lower panel: Coomassie staining of the GST fusion proteins after elution from the beads.