Extended Data Fig. 7: Substrate and its interaction residues in the Tim17-Mgr2 pathway.
From: Dynamic TOM–TIM23 supercomplex directs mitochondrial protein translocation and sorting
a, Mapping of the substrate in the TIM23 complex. The substrate residues around the α1 and α2 regions of the B2 segments are mutated to Bpa. The supercomplexes are assembled in the yeast strain yLS420 transformed with the plasmids pLSP2-x that encode the photoactive substrates. The cells are subjected to UV irradiation, followed by Mgr2 pulldown. The substrate, Mgr2, and their crosslinking products are detected by immunoblotting against the StrepII tag (Mgr2) and Flag tag (substrate). Position 155 of the substrate outside the TIM23 complex is included as a negative control. b, Comparison of the substrate passing through the restrictions of Tim17-Mgr2 (left) and the Sec channel (right, PDB ID: 6ITC). Tim17 and Mgr2 are colored brownish pink and gold, respectively. The N-half and C-half of the Sec channel are colored sky blue and plum, respectively. The restriction residues are shown as balls and sticks with those from Tim17-Mgr2 in crystal blue and those from Sec in red. The substrate density is colored green. The distances between the restriction residues and the centers of the substrate density are labeled. c, Spot assays to examine the growth of yeast strains with point mutations of the substrate-interacting residues of Tim17. d, Same as c except with additional Mgr2 knockout. e, Tim17 mutant selection on 5-FOA plates. The yeast strains, yLS270, yLS280, and yLS290, are transformed with Tim17 mutation plasmid pLSA4-X and selected on 5-FOA plates. The mutants that could not grow during selection are shown. f, Import of the metabolite carrier GGC1 via the TIM22 pathway into mitochondria with mutations of the restriction residues in Tim17. The imported and correctly folded GGC1 is detected as a ~ 100 kDa band in BN-PAGE 78.
