Figure 2.

Physical interactions between Cdc5p and Bbp1p. (A) Two-hybrid assays were conducted as described in MATERIALS AND METHODS with plasmids that expressed full-length or truncated forms of BBP1 or CDC5 as AD or DBD fusions, respectively. Cdc5pΔN/FAA possesses the FAA mutations, which disrupt the function of the polo-box domain of Cdc5p (Song et al., 2000). Numbers indicate the Miller units of β-galactosidase activity averaged from two independent experiments. Immunoblotting (bottom) indicated the expression levels of various constructs with Cdc28p as loading control. CDC5, pSK1405; CDC5ΔC, pSK1408; CDC5ΔN, pSK1390; CDC5ΔN/FAA, pSK1403; and BBP1 (1-385), pSK1883. (B and C) In vitro interaction studies were carried out using various full-length or truncated forms of GST-Bbp1p or GST-Mps2p as ligands. Sf9 cell lysates expressing recombinant His6-HA-Cdc5p-Flag (B) or bacterially expressed, purified, recombinant T7-Cdc5p-His6 (C) were incubated with various ligands as described in MATERIALS AND METHODS. After SDS-PAGE, the amounts of bound His6-HA-Cdc5p-Flag or T7-Cdc5p-His6 were determined by immunoblotting with either an anti-FLAG antibody (B, top) or an anti-T7 antibody (C, top), whereas the amounts of GST, GST-Bbp1p, or GST-Mps2p ligands were determined by Coomassie staining (B, bottom) or immunoblotting with an anti-GST antibody (C, bottom). Input, 5% of His6-HA-Cdc5p-Flag or T7-Cdc5p-His6 that was incubated with GST, GST-Bbp1p, or GST-Mps2p. 1–385, pKL2497; 1–295, pKL2498; 1–243, pKL2500; 243–385, pKL2499; 295–385, pKL2501; GST-Bbp1p, pKL2497; and GST-Mps2p, pKL2496.