FIG. 4.

Mapping of the CP-interacting domain of P2 with the yeast two-hybrid system. (A and B) Yeast strain HF7c was cotransformed with the plasmids encoding CP fused to Gal4AD (pGAD-CP) and P2 or modified P2 fused to the Gal4BD (pGBT-P2, pGBT-P2Δ1 to pGBT-P2Δ4, pGBT-P2M1 to pGBT-P2M19), and the transformants obtained were tested for β-galactosidase (β-gal.) activity. Mutations introduced in the P2 coding sequence of pGBT-P2 correspond to large deletions (A) and small deletions or amino acid substitutions (B). (A) P2 and truncated P2 forms are depicted schematically by open boxes, and Gal4BD is depicted by the interrupted black boxes. (B) Amino acid substitutions and small deletions in the C-terminal region of P2 fusion proteins are indicated by bold letters and bold hyphens, respectively. Numbers above the open boxes and the amino acid sequences refer to positions in P2. β-Galactosidase activity was determined by filter and liquid assay procedures. For the filter assay quantifications, β-galactosidase activity was assessed by the intensity of the blue color of yeast clones after 6 h: +++, dark blue; ++, intermediate color; +, light blue; +/− pale blue; −, no color. For the liquid-assay quantifications, the values indicate percent β-galactosidase activities relative to those obtained with the wild-type P2 construct, measured in extracts from yeast cells expressing AD-CP and one of the BD-P2 versions. Values given are the mean of the relative β-galactosidase activities (and their standard deviations) of five independent clones. The β-galactosidase activity of yeast clones harboring the P2 wild-type construct was, on average, 2.7 β-galactosidase units, as defined in Materials and Methods. The control corresponds to a yeast clone transformed with pGBT9 and pGAD424; n.d., not determined. (C) Role of the C-terminal residues of P2 in establishing the interaction with CP. Residues which are involved are shown in bold type. Boxed residues are crucial and underlined residues are important for the association with CP. The importance of residues QYK⁹⁷ was not assessed.