Identification of the kif1bβ molecular motor as a direct binding partner of Fignl1. (A) Schematic representation of zebrafish Fignl1 (Dr_Fignl1), mouse fignl1 (Ms_fignl1), and mouse kif1bβ (Ms_ kif1bβ). Specific domains are shown as boxes, and numbers indicate amino acids that delineate domain frontiers. EB1/3, EB-binding domain; AAA, ATPase domain; Motor, MT-binding domain; FHA, forkhead-associated domain; PH, Pleckstrin homology; Y2H, yeast two-hybrid screen. ATG1, ATG2, and ATG3 indicate alternative translation start sites identified in the zebrafish fignl1 sequence. Black arrows summarize the results of the yeast two-hybrid assay. (B and C) Ms_HA-fignl1 specifically associates with YFP-Hu_KIF1Bβ. Co-IP of Ms_HA-fignl1 and YFP-Hu_KIF1Bβ from protein extracts of COS-7 cells transfected with YFP-Hu_KIF1Bβ (B and C) or GFP (B) and Ms_HA-fignl1. (D and E) Co-IPs of Dr_Fignl1-HA (D) and Dr_Fignl1Δ1-173-HA (E) with YFP-Hu_KIF1Bβ. Co-IPs were performed from protein extracts of COS-7 cells transfected with YFP-Hu_KIF1Bβ and Dr_Fignl1-HA (D) or Dr_Fignl1Δ1-173-HA (E). (B–E) Immunoprecipitations were performed with GFP- (B, D, and E) or HA-trap antibodies (C). Immunoprecipitated and coimmunoprecipitated proteins were revealed by Western blot using HA or GFP antibodies. In, Input; IP, immunoprecipitation; WB, Western blot; Rb, rabbit; m, mouse. (F) COS-7 cells transfected with YFP-Hu_KIF1Bβ and Dr_Fignl1-HA or Dr_Fignl1Δ1-173-HA and immunolabeled with HA, GFP, and tyrosinated tubulin (Tub) antibodies. Bottom panels are higher magnifications of the boxed regions in the corresponding upper panels. White arrows in the right-hand panels indicate colocalization events that occur along MTs. Scale bars, 20 µm for upper panels and 5 µm for lower panels.