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. 2020 Oct 29;18:156. doi: 10.1186/s12915-020-00888-z

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© The Author(s) 2020

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Binding of [³⁵S]-labeled polypeptides to the purified cytosolic domain of yeast Tom70 and to isolated mitochondria. a SDS-PAGE of 0.75 μg isolated Tom70_cd (cytosolic domain, residues 38–617). b Binding of [³⁵S]-labeled polypeptides to Tom70_cd. Purified Tom70_cd was pre-bound to Ni-NTA beads and incubated with reticulocyte lysate containing radiolabeled wild type AAC (AAC_WT), or different segments of the AAC fused to DHFR: C-terminal part of AAC module III including the matrix loop and helix 6 (ML3-TMD6, residues 238–313), the matrix loop (ML3, residues 238–277), helix 6 (TMD6, residues 277–313), or the AAC derivative lacking the positively charged residues in the matrix loops (AAC_Δpos). Bound radiolabeled proteins were quantified using the phosphorimager, SD, n = 3. c Binding of [³⁵S]-labeled AAC_WT to isolated Tom70_cd pretreated with N-ethylmaleimide (NEM). The assays were performed as in b, but the Ni-NTA-bound Tom70_cd was preincubated with up to 4 mM NEM as indicated. The amount of bound protein in absence of NEM was set to 100%. SD, n = 4. d Binding of [³⁵S]-labeled AAC_WT to mutant versions of Tom70_cd. Single cysteines in positions 439, 450, or 141, respectively, were exchanged against alanine. The modified Tom70_cd proteins were bound to Ni²⁺-NTA, and as indicated, some samples were treated with 2 mM NEM. All beads were washed with assay buffer and subsequently used for binding assays as in c. SD, n ≥ 6. e Location of cysteine residue C141, arginine R171, and serine S174 in Tom70. The structural data were taken from Wu and Sha [53], PDB 2GW1. f Binding of [³⁵S]-labeled yeast AAC2 to isolated mitochondria containing wild type Tom70. Radiolabeled AAC2 was synthesized in reticulocyte lysate and preincubated with apyrase; the mitochondria were pretreated with 2 mM NEM (+ NEM) or left untreated (− NEM). Binding in absence of NEM within 12 min was set to 100%, SD, n = 3. g Binding of [³⁵S]-labeled AAC2 to mitochondria isolated from a yeast strain expressing Tom70_C141A. The assay was carried out as in f. Binding in absence of NEM within 12 min was set to 100%, SD, n = 3. h Binding of AAC to mitochondria pretreated with NEM and trypsin, SD, n = 3