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. 2023 Aug 1;621(7979):627–634. doi: 10.1038/s41586-023-06477-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a, ColabFold structural protein complex model of Tim17 (tan) and Tim23 (grey) heterodimers. Tan, endogenous intramolecular disulfide bond formed between Tim17 Cys10 (C10) and Cys77 (C77); orange, Tim17 Cys16 (N16C) and Tim23 Cys98 (C98) forming an intermolecular disulfide bond. b, In organello disulfide bond formation between Tim17 and Tim23. Mitochondria isolated from yeast strains expressing SCF Tim17 or Tim17 N16C were left untreated or treated with the reductant dithiothreitol (DTT; red) or oxidant 4,4′-dipyridyl disulfide (4-DPS; Oxid.) followed by SDS–PAGE. Tim17-S-S-Tim23, disulfide-linked Tim17 and Tim23. *Unidentified Tim17-specific conjugate. c, Import of Jac1–sfGFP into SCF Tim17 or N16C mitochondria followed by oxidation with 4-DPS. Jac1–sfGFP-containing TOM–TIM23 supercomplexes were isolated with a GFP nanobody. Load (1%) and eluate (100%) fractions were analysed by SDS–PAGE and immunodecoration. d,e, Import of Jac1–sfGFP into wild-type or Tim17^2xStrep (d) and ^HisSumo*Tim23 mitochondria (e). TIM23 complexes were isolated by tandem purification first using streptavidin (Strep; d) or immobilized metal affinity chromatography (IMAC; e) followed by purification of Jac1–sfGFP-containing TOM–TIM23 supercomplexes using a GFP nanobody. Fractions were analysed by SDS–PAGE and immunodecoration. Load, 0.25%; first elut., first elution (2.5%); second elut., second elution (100%).