Extended Data Fig. 8: Generation of stalled translocation intermediate complexes of TIM23–TOM–substrate and probing the preprotein translocation path by UV photocrosslinking.

a, Formation of a supercomplex containing TIM23, TOM, and the Grx5-S80-sfGFP substrate (S80 is a hydrophilic segment derived from yeast Cyb2) was confirmed by BN-PAGE. Where indicated, expression of Grx5-S80-sfGFP (C-terminally ALFA-tagged) was induced. After solubilizing mitochondria with digitonin, ALFA-tag immunoprecipitation (IP) was performed, and eluates were subjected to BN-PAGE and analyzed by immunoblotting (IB). Where indicated, eluate was treated with additional dodecylmaltoside (DDM) to dissociate the TIM23 complex. We note that while a substantial population of Grx5-S80-sfGFP stalls in TOM and TIM23, a major population is fully imported into the matrix without stalling. Faster migration of the TOM-substrate band in the presence of DDM is likely due to changes in micelle properties⁸⁶ or partial dissociation of Tom subunits. b, Testing tagging of Mgr2. To enable detection of Mgr2 in immunoblots, an N-terminal Strep-tag was added to Mgr2. Temperature sensitivity^14,37 of an Mgr2-deleted strain (mgr2Δ) and strains additionally expressing Strep-Mgr2 was tested (all in a W303-1A background). Where indicated, cells were transformed with a plasmid encoding non-tagged Mgr2 under a native MGR2 promoter, or N-terminally Strep-tagged Mgr2 under the MGR2 promoter or an ALD6 promoter. Note that lesser growth rescue by MGR2 promoter-driven Strep-Mgr2 might be due to a reduced expression level by insertion of the Strep-tag at the N-terminus or partial impairment of the function of Mgr2. The partial rescue could be overcome by stronger expression of Strep-Mgr2 under the ALD6 promoter. All subsequent experiments were performed with the Strep-Mgr2 (pALD6) strain. c, As in b, but yeast whole cell lysates (from equal OD₆₀₀) were analyzed by immunoblotting to test expression of Strep-Mgr2. Asterisk, a putative precursor form of Mgr2 (ref. ⁸⁷). d, Co-immunoprecipitation of TIM23-TOM-substrate supercomplexes with and without Mgr2. The upper panel depicts two different stalling substrates used. Grx5-S99(TM)-sfGFP contains a 99-amino-acid-long segment with a TM helix between 36 and 51 amino acid residues (derived from Cyb2). Upon stalling, this TM is expected to be released to the IM. In lower panels, cells were induced for expression of the indicated substrate, and the mitochondrial lysates were subjected to ALFA-tag IP. Where indicated, the strain lacked endogenous Mgr2 (Δ) and/or expressed exogeneous Strep-tagged Mgr2. Asterisk, putative precursor of Mgr2. We note that Grx5-S80-sfGFP reproducibly showed reduced copurification of Tom40 in the absence of Mgr2. Although the cause of this decrease is unclear, it is unlikely due to impaired presequence engagement of Mgr2-lacking TIM23 since we did not observe a similar decrease with Grx5-S99(TM)-sfGFP. e, Photocrosslinking with Bpa-incorporated Grx5-S80-sfGFP. As in Fig. 3c, but showing the immunoblots for Tim44, Tom22, Tom40, and the substrate. f, Verification of a covalent linkage formed between Grx5-S80-sfGFP and the indicated TIM23 subunit upon photocrosslinking. Beads were split to two halves after a regular wash step of IP, and one of them was additionally washed twice with 6 M urea to remove proteins that are noncovalently associated to the substrate (note that ALFA-tagged protein retains to ALFA-nanobody beads in this denaturing condition⁸⁸). Asterisk, putative partial degradation products. g, The crosslinked band (red arrowhead) between Grx5-S80-sfGFP (Bpa in position 31 of S80) and Tim17 was verified with successive IPs using the ALFA-tag and HA-tag. After the first ALFA-tag IP, proteins were denatured by SDS. Black arrowhead, the uncrosslinked substrate; asterisk, a putative partial degradation product of the crosslinked species. h and i, The crosslinked band (red arrowheads) between Grx5-S80-sfGFP and Mgr2 (h) or Tim44 (i) was verified by SDS-denatured pulldown (for Strep-Mgr2) or IP (for Tim44). Bpa sites are located in indicated positions of S80. Black arrowhead, the uncrosslinked substrate; hash, putative cross-reacted IgG. Data in a–d and f–i are representative of two independent experiments. Data in e is representative of three independent experiments.