Mapping the interacting domains of Bem1p and Exo70p. (A) The PX and PB1 domains of Bem1p are each necessary and together sufficient for interaction with Exo70p. At the upper left is a schematic diagram of the Bem1p protein with SH3-1, SH3-2, PX, and PB1 domains highlighted. In the lower left part, each bar represents either full-length Bem1p or a fragment. Each purified His₆-tagged Bem1p fragment was incubated with glutathione beads with prebound GST-Exo70p or control protein GST-Sec22NTp. Bound Bem1p fragments were detected with anti-His antibody (right). (B) Exo70p does not affect the interaction between Cdc24p and Bem1p. GST-tagged Bem1p or control protein Gea2Cp were immobilized on glutathione beads and then incubated with purified His₆-MBP-Cdc24C in the presence or absence of purified His₆-Exo70p. Bound proteins were detected as described in the Materials and methods. (C) Cdc24p and Exo70p can form a ternary complex with Bem1p. GST-Exo70p was immobilized on glutathione beads and then incubated with purified His₆-MBP-Cdc24C in the presence or absence of purified His₆-Bem1p. Bound proteins were detected as described in the Materials and methods. (D) Each helical bundle domain of Exo70p contributes to the interaction with Bem1p. GST-tagged full-length or domain-deleted Exo70p proteins were immobilized on glutathione beads and then incubated with Bem1p-His₆. Bound proteins were detected as described in the Materials and methods.