Figure 5.

NIX1 contains a minimal receptor interacting domain spanning 39 amino acids. EMSAs were performed by incubating a ³²P-labeled GALp response element with Gal4-RORβ_201–459 or Gal4-RORβ_201–440. Receptor-DNA complexes were challenged with purified GST, GST-NIX1, or various GST-NIX1 deletion mutants. (A) GST-NIX1 specifically interacts with DNA-bound Gal4-RORβ_201–459, but does not bind to DNA alone or a DNA/Gal4-RORβ_201–440 complex. Both GST-NIX1 amino acids 1–99 and GST-NIX1 amino acids 61–99 bind the receptor-DNA complex and mediate ternary complex formation comparable to GST-NIX1. GST alone, GST-NIX1 amino acids 1–60, or GST-NIX1 amino acids 100–229 do not associate with DNA-bound RORβ. (B) Gal4-RORβ_201–459 bound to a ³²P-labeled GALp oligonucleotide, was incubated with equal amounts of purified recombinant GST-NIX1 fusion proteins. Resulting protein-DNA complexes were separated on nondenaturing 6% polyacrylamide gels. GST-NIX1 and GST-NIX1 amino acids 61–99 specifically interact with DNA-bound RORβ. Replacement of both L87 and L88 with A in either full-length NIX1 or in NIX1 amino acids 61–99 resulted in proteins GST-NIX1 (AA) or GST-NIX1 amino acids 61–99 (AA), which do not associate with receptor-DNA complexes.