Fig. 2. Qualitative two-hybrid interaction assays in yeast reveal domains required for interactions between Ssdp proteins and Ldb/Chip proteins from mice and flies. Schematic diagram of recombinant proteins fused to the DNA-binding domain (DBD) or activation domain (AD) of GAL4. Mouse Ldb1 and Drosophila Chip are depicted in white, mouse Ssdp2 and Drosophila Ssdp in gray, and mouse Lhx3 as a positive control in black. Interactions between proteins were measured by ß-galactosidase activity and were scored as either positive (+) or negative (-). (A) Sequences between amino acids 201 and 255 of Ldb1 are required for interaction with Ssdp2. (B) The N-terminal 100 amino acids of Ssdp2 are sufficient for interaction with Ldb1. (C) Upon switching the configuration of the fusion proteins, the requirements of amino acids 1-100 of Ssdp2 and 201-255 of Ldb1 are reiterated, supporting the specificity of the interaction. (D) Further refinement of Ldb1 sequences required for interaction with Ssdp2 through two deletions of 10 amino acids each. Deletion of amino acids 214-223 disrupts the interaction with Ssdp2, but has no effect on the ability of Ldb1 to bind the LIM domains of Lhx3. (E) The Drosophila melanogaster (D.m.) orthologs Ssdp and Chip give similar results to those obtained for the mouse proteins. Ssdp amino acids 1-98 are sufficient for interaction with Chip, and removal of amino acids 387-426 of Chip prevents binding to Ssdp but not to the Lhx3 LIM domains.

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