Fig. 3. Tou physically interacts with Pnr. (A) Structural features of the Pnr and Tou proteins used in the present study. The DNA binding domain of Pnr (Pnr^DBD), containing the two zinc fingers (grey boxes) and the C-terminal domain (Pnr^CT) containing the two amphipathic -helices (black boxes). The mutations associated with pnr^D1 and with pnr^VX1 are localized within the N-terminal zinc finger and in the N terminus of the helices respectively. A schematic drawing of the Tou domains used throughout this study (TouA to TouK). The functional domains of Tou are schematized as in Fig. 1. (B) Tou physically interacts with Pnr in yeast, through both the DBD and the C-terminus of Pnr. Expression vectors for unfused LexA^DBD(-) or LexA^DBDPnr^DBD, LexA^DBDPnr^CT, LexA^DBDPnr^WT, LexA^DBDPnr^D1, LexA^DBDPnr^VX1 were introduced into L40 cells together with the unfused VP16^AD (not shown) or VP16^ADTouA. Protein extracts made from cultured L40 transformants were assayed for ß-galactosidase activity (expressed in nanomoles of substrate/mn/mg of protein). Values (±10%) are the averages of three independent experiments. (C,D) Tou physically interacts with (C) full-length Pnr, (D) the DBD of Pnr and the C terminus (CT) of Pnr in transfected cells. In each case, an immunoblot of a representative set of transfected cell extracts is shown. The transfected expression vectors are shown at the top of the panels. The B10 monoclonal antibody used to immunoprecipitate the extracts is shown on the left of the panels. The antibodies used to reveal the blots are indicated at the bottom. Pnr is recognized by the 2B8 antibody, GSTPnr^DBD and GSTPnr^CT are detected by the D10 antibody and the B10-tagged TouA is recognized by the B10 antibody. The locations of the proteins including the immunoglobulin heavy chain [IgG(H)] are indicated at the sides. (E) Tou directly interacts both with the DBD and the CT of Pnr in vitro. (E) Autoradiographs of SDS-PAGE gels from representative affinity chromatography experiments performed with GST control beads (lane 2), GSTPnr^DBD beads (lane 3) and GSTPnr^CT beads (lane 4) and in vitro translated ³⁵S proteins as indicated on the left. One-tenth of the ³⁵S input is shown in lane 1. Luciferase is used as a negative input. Experiments were performed three times and, with all proteins except luciferase, 50-fold more protein bound to GSTPnr^DBD and GSTPnr^CT than to GST control. (F) The N terminus of the MBD domain of Tou mediates interaction with Pnr. Expression vectors for unfused LexA^DBD (not shown) or LexA^DBDPnr^WT were introduced in L40 cells together with the unfused VP16^AD (not shown) or VP16^ADTouA, VP16^ADTouC, VP16^ADTouD, VP16^ADTouJ or VP16^ADTouK. Protein extracts made from cultured L40 transformants were assayed for ß-galactosidase activities (expressed in nanomoles of substrate/mn/mg of protein). Values (±10%) are the averages of three independent experiments.