The interaction between Bcp1 and uL14 is important to release uL14 from Kap. (A) The N-terminus (magenta) is shown on the Bcp1 structure. (B) The diagram of Bcp1 shows the N terminus and NLS sequences. The mutation site of bcp1ts is F241S. (C)BCP1, bcp1ΔN10, bcp1ΔN20, and bcp1ΔN40 were transformed to bcp1ts and applied in the growth test. (D) The localization of Bcp1-GFP, bcp1ΔN10-GFP, bcp1ΔN20-GFP, and bcp1ΔN40-GFP were examined under fluorescence microscopy. (E) GST-Kap121 was immobilized on the glutathione beads and interacted with purified Bcp1, bcp1ΔN10, bcp1ΔN20, and bcp1ΔN40 (left panel). The positions of Bcp1 were indicated with asterisk (*), and the interaction signals were also detected with α-Bcp1 antibody. In parallel, bcp1ΔN mutants were immobilized on the NTA beads, and the interactions with uL14 were also examined (middle panel). The position of uL14 was indicated with asterisk (*) and the interaction signals were also detected with α-uL14 antibody. The purified Bcp1 and bcp1ΔN mutant proteins were shown (right panel, input). (F) GST-Kap121 in complex with uL14 or uL14∆loop was immobilized on the glutathione beads. Purified Bcp1, bcp1ΔN10, or bcp1ΔN20 was added and incubated for another hour at 4°C. The partitions of uL14 on the beads and in the supernatants were analyzed. The uL14 signals in the supernatants were detected by western blotting. (G) Kap121-TAP and Kap123-TAP were immunopurified in WT with additional expressions of uL14 or uL14∆loop. The associated proteins were probed with anti-Rkm1 and anti-HA antibodies. Source data are available for this figure: SourceData F5.