Figure 4: TRAF3IP1 mutations impair folding of the CH domain and interaction with MAP4.
From: Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization
(a) Crystal structure of the CH domain of MmIFT54 (based on PDB entry 2EQO) showing that the I17 and V125 residues locate in conserved hydrophobic pockets (dotted line circles). The mutant residues S17, A125 and M125 were introduced (red) to show their effects on these hydrophobic pockets. (b) Lysates from HEK293T cells co-expressing Flag-tagged WT or mutant forms of MmIFT54 (p.K155*, p.I453R and p.M458Mfs3* correspond to the human mutations p.R155*, p.M520R and p.M525Mfs3*) and GFP-MAP4 were immunoprecipitated with an anti-GFP antibody. The co-immunoprecipitation of GFP-MAP4 and Flag-IFT54 constructs was followed by western blot (WB) using GFP and Flag antibodies. (c) Serum-starved fibroblasts were fixed in PFA to visualize ciliary MAP4 (red; acetylated α-tubulin, green). Scale bar, 2 μm. (d) Intensity of ciliary MAP4 staining (mean ± s.e.m. of n=5 experiments (that is ≈150 cilia, *P<0.05, ***P<0.001, Dunn's post-hoc test).
