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. 2018 Jan 3;27(5):837–852. doi: 10.1093/hmg/ddy004

Figure 4.

Figure 4.

The LYR motif of CIAO1 mediates its binding to HSC20, which is essential for formation of the ISC/CIA complex. (A) In the CIAO1 primary sequence, the canonical LYR motif and the LYR-like sequences are highlighted with colored bars above the motifs. (B) IP of endogenous C-HSC20 from cytosolic fractions of cells stably expressing CIAO1-V5 WT or the mutants, as indicated. Eluates after IP of C-HSC20 were analyzed by western blot to check for the interaction between CIAO1-V5 WT or mutants and HSC20 (superscript D, T and Q designate the double, triple and quadruple CIAO1 mutants in which substitution of the LYR motif was combined with mutagenesis of one or more LYR-like sequences, as indicated. Empty-V5 is a control cell line, which was stably transfected with the empty vector). (C) Pull-down assays of 35S-labeled CIAO1-V5 WT or its mutants, as indicated, were performed in the presence of HSC20-FLAG/MYC (CIAO1IWK-V5 is the mutant in which I87W88K89 was replaced by alanines). (D) IPs of CIAO1-V5 WT or the L176Y177R178 to triple alanine mutant, followed by IBs to V5, CIAO1 and the Fe-S proteins ELP3 and ERCC2. The recipient proteins ELP3 and ERCC2 did not coprecipitate with CIAO1 when the LYR motif of CIAO1 was mutagenized. V5 IPs of CIAO1 WT (E) or the LYR mutant (F), followed by native and 2D-SDS-PAGE resolution of the competitively eluted cytosolic complexes. IBs to CIAO1, HSC20, HSPA9 and the Fe-S protein ERCC2 are shown (B–F, n = 4 biological replicates). See also Supplementary Material, Figure S4.