Fig. 2.

dCRY interacts with INAD. (A) Coimmunoprecipitation and Western blot confirming the interaction between HACRY and INAD in flies overexpressing HACRY (yw;tim-GAL4/+; UAS-HAcry/+). tim-GAL4 flies were used as control (“C”). Heads were collected as in Fig. 1B. Membranes were probed with anti-INAD and anti-HA antibodies. inaD¹ and w¹¹¹⁸ flies, collected at ZT1, were used as negative and positive controls of the antibody, respectively. (B) Identification of the interaction domains of dCRY and INAD using the yeast two-hybrid system. The five INAD PDZ domains are shown where modeled and assigned to putative PDZ subtypes depending on the residue types at the peptide-binding site. Relevant sequence motifs are shown as empty rectangles in the INAD and CRY sequence diagrams. Different domains of INAD were tested for interaction with the full-length dCRY in the presence of light, and different domains of dCRY were tested for interaction with the full-length INAD under both light and dark conditions (open and filled bars, respectively). Interacting fusions are shown in black, and relative β-galactosidase activity (Miller units) is reported for each fusion. Mean ± SEM of at least seven independent clones for each fusion, analyzed in triplicates, is shown. An extended version of the PDZ2–3 tandem, INAD (207–448), exhibits a significantly stronger affinity for dCRY compared with the whole protein (F_14,87 = 67.81, P < 0.0001). The interaction between dCRY and INAD occurred in a light-dependent fashion with the C terminus of dCRY being crucial. On the other hand, these last 22 amino acids of the protein showed a light-independent affinity for INAD with a significantly stronger interaction in the light compared with the dark (t₁₃ = 2.6, P = 0.02). (C) Yeast two- and three-hybrid assays highlighting that the interaction between dCRY and NINAC is mediated by INAD. The schematic shows the different proteins used as bait or prey fusion: C, dCRY; N, NINAC; I, INAD. Relative β-galactosidase activity (Miller units) is reported for each fusion. Mean ± SEM of at least six independent clones for each fusion, analyzed in triplicates, is shown. The expression of dCRY and NINAC alone does not result in the activation of the reporter gene. The expression of INAD in the yeast nucleus, to generate a three-hybrid system, shows that INAD acts as a structural bridge (BRIDGE) between the two proteins (F_3,24 = 57.20, P < 0.0001). The interactions of dCRY–INAD and INAD–NINAC are also shown.