Fig. 1: The architecture of substrate-engaged TOM–TIM23 supercomplex.

a Schematic diagram of the TOM–TIM23–Jac1^sfGFP supercomplex capture. b Blue native PAGE analysis of the TOM–TIM23–Jac1^sfGFP supercomplex. c Architecture of the TOM–TIM23–Jac1^sfGFP supercomplex. d, e Structure of the Jac1^sfGFP-engaged TOM complex. f Spot assays of tom5Δ strain rescued by Tom5 truncations. g Steady-state level of the TOM complex corresponding to f. TOM′ representing the TOM complex lacking of Tom5. h, i Real-time import of the CytB₂ (220Δ19)–pep86 by NanoLuc assay. j Import assays of radiolabeled Jac1 into mitochondria under the indicated conditions. k Import assays of radiolabeled Jac1 into mitochondria which were preincubated with rabbit serum against the N-terminus of Tom5. In j, k data are means ± SEM representing at least three biologically independent samples. P-values were calculated by two-tailed Student’s t-test. Δφ, membrane potential; p, precursor at the receptor stage; p*, precursor at the post-receptor stage; m, mature Jac1. l Potential translocation path for presequence-carrying preproteins. The electrostatic potential surface of full-length Tom5 (predicted by AlphaFold2) was displayed. Red, negatively charged region. m Working model of translocation path of the presequence-carrying preproteins across the outer and inner membranes of mitochondria.