Effect of cofactors on FAC-RED interaction. (A) The FMN-binding domain of RED is required for interaction with FAC. Schematic diagram indicating functional domains of RED analyzed for binding to FAC in the yeast two-hybrid system. The intensity of blue color corresponding to β-galactosidase activity was assessed visually and scored as follows: minus, white; double plus, blue; triple plus, dark blue. Anc, membrane anchor. (B) Failure of FAC to bind RED in presence of cofactors. Radiolabeled lysates of COS-1 cells transfected with both FAC and RED were divided into equal volumes, immunoprecipitated sequentially with anti-FAC antibody and protein A-agarose, and analyzed by SDS-PAGE and autoradiography. Increasing amounts (0, 0.1 mmol/L, and 1.0 mmol/L) of FMN, FAD, or cytochrome c were added to otherwise identical lysates during immune complex formation.