Fig. 4. Tou physically interacts with Chip. (A) Structural features of the Chip proteins used in this study: the N-terminal homodimerization domain (DDT) of Chip (N^TChip; black box) and the C-terminal LIM-interacting domain (LID) of Chip (C^TChip; grey box). (B) Tou interacts with Chip in yeast through the N-terminal homodimerization domain. Expression vectors encoding the unfused LexA^DBD (-) or the LexA^DBDChip, the LexA^DBDN^TChip, the LexA^DBDC^TChip were introduced in L40 cells together with the unfused VP16^AD (not shown) or VP16^ADTouA. Protein extracts made from L40 transformants, grown in liquid medium, were assayed for ß-galactosidase activities, which are expressed as in Fig. 3B. (C) Tou interacts with Chip in transfected cells. The layout is as in Fig. 3C. The flagged Chip is detected with the M2 antibody whereas the B10-tagged proteins (B10-Chip, B10-N^TChip, B10-C^TChip) are recognized by the B10 antibody. (D) Tou directly interacts with Chip in vitro. (D) Autoradiographs of SDS-PAGE gels from representative affinity chromatography experiments performed with GST control beads (lane 2) and GST Chip beads (lane 3) and in vitro translated ³⁵S proteins as indicated on the left. One-tenth of the ³⁵S input is shown in lane 1. Luciferase was used as a negative input. Experiments were performed three times and 50-fold more ³⁵S labelled TouA bound to GST Chip than to GST control. (E) The DDT domain of Tou mediates interaction with Chip. Expression vectors encoding the unfused LexA^DBD (not shown) or the LexA^DBDChip were introduced into L40 cells together with the unfused VP16^AD (not shown) or the VP16^ADTouA, VP16^ADTouC, VP16^ADTouD, VP 16^ADTouL, VP16^ADTouM or VP16^ADTouN. Protein extracts made from L40 transformants, grown in liquid medium, were assayed for ß-galactosidase activities (expressed as in B).