Extended Data Fig. 6: Formation of the two exit sites of the TOM channel for different classes of precursor proteins.
From: Structure of the mitochondrial import gate reveals distinct preprotein paths
a, Disordered regions of Tom22 and Tom40 facing the IMS. b, The TOM complex viewed from the IMS side shows that the trans presequence binding site (orange) is formed by the hydrophilic loop of Tom7 (residues 43–49), the hydrophilic β1–β2 loop (residues 92–96, M94 is disordered) and C-terminal domain (residues 363–387, residues after L374 are disordered, G363–E366 form a loop and E367–G373 form a helix) of Tom40 and the acidic IMS domain of Tom22 (residues 120–152, residues after 131 are disordered, the transmembrane helix is extended up to F130). Disordered parts are shown with beads. c, Crosslinked products of Tom22 containing BPA at the indicated positions were detected by the indicated antibodies. Crosslinked products between Tom22 and Tim50 (22–50) are indicated. Representative data of two independent experiments. d, Radiolabelled Oxa1 precursor was imported into wild-type and Tom40(Δ364–387) mitochondria for the indicated periods in the absence or presence of a membrane potential (Δψ) and analysed by blue native electrophoresis and autoradiography. Representative data of three independent experiments. Quantification of Oxa1 bound to the TOM complex; mean values ± s.e.m. (n = 3 biologically independent experiments); the amount of bound Oxa1 in wild-type mitochondria after 20 min (−Δψ) was set as 100% (control). e, Mitochondria isolated from wild-type and Tom40(Δ364–387) yeast grown at 30 °C were analysed by SDS–PAGE and western blotting using the indicated antisera. Representative data of four independent experiments. Amount of total mitochondrial protein is indicated. f, Mitochondria isolated from wild-type and Tom40(Δ364–387) yeast grown at 30 °C were analysed by blue native electrophoresis and western blotting with Tom40 antiserum. Representative data of four independent experiments. g, Radiolabelled Tim9 was imported and assembled into the TIM22 complex in wild-type and Tom40(Δ364–387) mitochondria; data are mean ± s.e.m. (n = 3 biologically independent experiments); the amount in wild-type mitochondria after 40 min import was set as 100% (control). h, The trans presequence binding sites for presequence-containing proteins (b) and the N extension of Tom40 recruiting small TIM chaperones (for presequence-lacking β-barrel and carrier proteins) and Mia40 (for MIA substrates) are spatially separated to form distinct precursor exit sites at the outlet of the TOM channel. The two exit sites are connected to the different translocation paths (yellow broken lines) that include either the aligned acidic patches (red) or the aligned hydrophobic patches (green) inside the Tom40 β-barrel pore (Extended Data Fig. 9). Disordered parts are shown with beads.
