Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A multipoint guidance mechanism for β-barrel folding on the SAM complex

Nature Structural & Molecular Biology volume 30, pages 176–187 (2023)Cite this article

Subjects

An Author Correction to this article was published on 25 January 2023

This article has been updated

Abstract

Mitochondrial β-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic β-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last β-strand (β-signal) of the substrate—the 19-β-stranded Tom40 precursor—to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40’s first β-segment (β1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces β1 to promote its pairing with Tom40’s last β-strand to complete barrel formation with the assistance of Sam37’s dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial β-barrel folding.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structure of Tom40G354A complexed with the SAM complex as the SAMTom40-early intermediate.
Fig. 2: Hybrid β-barrel of SAMTom40-early intermediate.
Fig. 3: Directional β-barrel folding of Tom40.
Fig. 4: Exchange of the β1 segment with the N region of Tom40 inside the growing β-barrel lumen.
Fig. 5: Role of the Tom40 N region in β-barrel formation.
Fig. 6: Role of Sam37 in β-barrel formation.
Fig. 7: β-Barrel formation under multipoint guidance by the SAM complex.

Similar content being viewed by others

Data availability

The cryo-EM density map and atomic coordinates of the yeast SAMTom40-early complex have been deposited to the Electron Microscopy Data Bank and Protein Data Bank with accession numbers EMD-32019 and 7VKU, respectively. Source data are provided with this paper.

Change history

References

  1. Neupert, W. & Herrmann, J. M. Translocation of proteins into mitochondria. Ann. Rev. Biochem. 76, 723–749 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Walther, D. M. & Rapaport, D. Biogenesis of mitochondrial outer membrane proteins. Biochim. Biophys. Acta 1793, 42–51 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Walther, D. M., Rapaport, D. & Tommassen, J. Biogenesis of β-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence. Cell. Mol. Life Sci. 66, 2789–2804 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hagan, C. L., Silhavy, T. J. & Kahne, D. β-Barrel membrane protein assembly by the Bam complex. Annu. Rev. Biochem. 80, 189–210 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Wu, R., Stephenso, R., Gichaba, A. & Noinaj, N. The big BAM theory: an open and closed case? Biochim. Biophys. Acta 1862, 183062 (2020).

    Article  CAS  Google Scholar 

  6. Höhr, A. I. C., Straub, S. P., Warscheid, B., Becker, T. & Wiedemann, N. Assembly of β-barrel proteins in the mitochondrial outer membrane. Biochim. Biophys. Acta 1853, 75–88 (2015).

    Google Scholar 

  7. Selkrig, J. et al. Discovery of an archetypal protein transport system in bacterial outer membranes. Nat. Struct. Mol. Biol. 19, 506–510 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Paschen, S. A. et al. Evolutionary conservation of biogenesis of β-barrel membrane proteins. Nature 426, 862–866 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Wiedemann, N. et al. Machinery for protein sorting and assembly in the mitochondrial outer membrane. Nature 424, 565–571 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Gentle, I. et al. The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol. 164, 19–24 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ishikawa, D., Yamamoto, H., Tamura, Y., Moritoh, K. & Endo, T. Two novel proteins in the mitochondrial outer membrane mediate β-barrel protein assembly. J. Cell Biol. 166, 621–627 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Waizenegger, T. et al. Tob38, a novel essential component in the biogenesis of β-barrel proteins of mitochondria. EMBO Rep. 5, 704–709 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Klein, A. et al. Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane. J. Cell Biol. 199, 599–611 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yamano, K., Tanaka-Yamano, S. & Endo, T. Mdm10 as a dynamic constituent of the TOB/SAM complex directs coordinated assembly of Tom40. EMBO Rep. 11, 187–193 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ellenrieder, L. et al. Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10. Nat. Commun. 7, 13021 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Diederichs, K. A. et al. Structural insight into mitochondrial β-barrel outer membrane protein biogenesis. Nat. Commun. 11, 3290 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Takeda, H. et al. Mitochondrial sorting and assembly machinery operates by β-barrel switching. Nature 590, 163–169 (2021).

    Article  CAS  PubMed  Google Scholar 

  18. Wang, Q. et al. Structural insight into the SAM-mediated assembly of the mitochondrial TOM core complex. Science 373, 1377–1381 (2021).

    Article  CAS  PubMed  Google Scholar 

  19. Kutik, S. et al. Dissecting membrane insertion of mitochondrial β-barrel proteins. Cell 132, 1011–1024 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Becker, T. et al. Assembly of the mitochondrial protein import channel. Role of Tom5 in two-stage interaction of Tom40 with the SAM complex. Mol. Biol. Cell 21, 3106–3113 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wenz, L.-S. et al. Sam37 is crucial for formation of the mitochondrial TOM–SAM supercomplex, thereby promoting β-barrel biogenesis. J. Cell Biol. 210, 1047–1054 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Stroud, D. A. et al. Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex. Mol. Biol. Cell 22, 2823–2832 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Höhr, A. I. C. et al. Membrane protein insertion through a mitochondrial β-barrel gate. Science 359, 6834 (2018).

    Article  Google Scholar 

  24. Araiso, Y. et al. Structure of the mitochondrial import gate reveals distinct preprotein paths. Nature 575, 395–401 (2019).

    Article  CAS  PubMed  Google Scholar 

  25. Tucker, K. & Park, E. Cryo-EM structure of the mitochondrial protein-import channel TOM complex at near-atomic resolution. Nat. Struct. Mol. Biol. 26, 1158–1166 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Qiu, J. et al. Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation. Cell 154, 596–608 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Arnold, T., Poynor, M., Nussberger, S., Lupas, A. N. & Linke, D. Gene duplication of the eight-stranded β-barrel OmpX produces a functional pore: a scenario for the evolution of transmembrane β-barrels. J. Mol. Biol. 366, 1174–1184 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Remmert, M., Biegert, A., Linke, D., Lupas, A. N. & Söding, J. Evolution of outer membrane β-barrels from an ancestral ββ hairpin. Mol. Biol. Evol. 27, 1348–1358 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Jores, T. et al. Characterization of the targeting signal in mitochondrial β-barrel proteins. Nat. Commun. 7, 12036 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Jores, T. et al. Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial β-barrel proteins. J. Cell Biol. 217, 3091–3108 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chan, N. C. & Lithgow, T. The peripheral membrane subunits of the SAM complex function codependently in mitochondrial outer membrane biogenesis. Mol. Biol. Cell 19, 126–136 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Doyle, M. T. & Barnstein, H. D. Bacterial outer membrane proteins assemble via asymmetric interactions with the BamA β-barrel. Nat. Commun. 10, 3358 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Tomasek, D. et al. Structure of a nascent membrane protein as it folds on the BAM complex. Nature 583, 473–478 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wu, R. et al. Plasticity within the barrel domain of BamA mediates a hybrid-barrel mechanism by BAM. Nat. Commun. 12, 7131 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Xiao, L. et al. Structures of the β-barrel assembly machine recognizing outer membrane protein substrates. FASEB J. 35, e21207 (2021).

    Article  CAS  PubMed  Google Scholar 

  36. Doyle, M. T., Jimah, J. R., Hinshaw, J. E. & Bernstein, H. D. Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding. Cell 185, 1143–1156 (2022).

    Article  CAS  PubMed  Google Scholar 

  37. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Mastronarde, D. N. et al. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  39. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D 74, 814–840 (2018).

    Article  CAS  Google Scholar 

  45. Kim, D. N., Gront, D. & Sanbonmatsu, K. Y. Practical considerations for atomic structure modeling with cryo-EM maps. J. Chem. Inf. Model. 60, 2436–2442 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

  47. Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Tama, F. et al. Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM. J. Struct. Biol. 147, 315–326 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).

    Article  CAS  PubMed  Google Scholar 

  50. Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 54, 5.6.1–5.6.37 (2016).

    PubMed  Google Scholar 

  51. Wu, E. L. et al. CHARMM-GUI membrane builder toward realistic biological membrane simulations. J. Comput. Chem. 35, 1997–2004 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).

    Article  CAS  PubMed  Google Scholar 

  53. Jorgensen, W. L. et al. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  54. Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

    Article  Google Scholar 

  55. Roe, D. R. & Brooks, B. R. A protocol for preparing explicitly solvated systems for stable molecular dynamics simulations. J. Chem. Phys. 153, 054123 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Balusek, C. et al. Gumbart, accelerating membrane simulations with hydrogen mass repartitioning. J. Chem. Theory Comput. 15, 4673–4686 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577 (1995).

    Article  CAS  Google Scholar 

  58. McGibbon, R. T. et al. MDTraj: a modern open library for the analysis of molecular dynamics trajectories. Biophys. J. 109, 1528–1532 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Sastry, G. M. et al. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221–234 (2013).

    Article  PubMed  Google Scholar 

  60. Li, J. et al. The VSGB 2.0 model: a next generation energy model for high resolution protein structure modeling. Proteins 79, 2794–2812 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Lomize, M. A. et al. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res. 40, D370–D376 (2012).

    Article  CAS  PubMed  Google Scholar 

  62. Harder, E. et al. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 12, 281–296 (2016).

    Article  CAS  PubMed  Google Scholar 

  63. Kabsch, W., & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).

    Article  CAS  PubMed  Google Scholar 

  64. Struyvé, M., Moons, M. & Tommassen, J. Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J. Mol. Biol. 218, 141–148 (1991).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the members of the T.E. and N.P./N.W. laboratories for discussion and critical comments on the manuscript. This work was supported by JSPS KAKENHI to T.E. (15H05705 and 2222703), K.I. (16K21680, 18K11543 and 21H03551) and H.T. (18K14640), by a JST CREST grant to T.E. (JPMJCR12M1) and an AMED CREST grant to T.E. (21gm1410002h0002), by Deutsche Forschungsgemeinschaft (German Research Foundation) grants to T.B. (BE 4679/2-2; SFB1218 project ID 269925409), N.P. (PF 202/9-1; project ID 394024777) and N.W. (WI 4506/1-1 (project ID 406757425) and SFB 1381 (project ID 403222702)), Germany’s Excellence Strategy to N.P. and N.W. (CIBSS-EXC2189; Project ID 390939984) and European Research Council Consolidator grant number 648235 to N.W. The following grants are also acknowledged: a grant from the Takeda Science Foundation (to T.E.) and grants from the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED under grant numbers JP19am01011115 (to M.K.), JP21am0101114 (to K.I., C.M. and T.H.) and JP21am0101110 (support number 1976; to K.T). Technical assistance by J. Suzuki (T.E. laboratory) is acknowledged. H.T. was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science (18J00358) and is grateful to M. Shirouzu (RIKEN Institute) for the encouragement to H.T. I.G. was supported by an Alexander von Humboldt Foundation Research Fellowship.

Author information

Author notes

  1. Hironori Takeda

    Present address: Nara Institute of Science and Technology, Ikoma, Japan

  2. Lena-Sophie Wenz

    Present address: Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany

Authors and Affiliations

  1. Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan

    Hironori Takeda & Toshiya Endo

  2. Institute of Biochemistry and Molecular Biology, Centre for Biochemistry and Molecular Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Jon V. Busto, Caroline Lindau, Iniyan Ganesan, Lena-Sophie Wenz, Nikolaus Pfanner & Nils Wiedemann

  3. Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Akihisa Tsutsumi & Masahide Kikkawa

  4. Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan

    Kentaro Tomii & Yu Yamamori

  5. Cellular and Molecular Biotechnology Research Institute, AIST, Tokyo, Japan

    Kenichiro Imai, Takatsugu Hirokawa & Chie Motono

  6. Division of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Takatsugu Hirokawa

  7. Transborder Medical Research Center, University of Tsukuba, Tsukuba, Japan

    Takatsugu Hirokawa

  8. Computational Bio Big-Data Open Innovation Laboratory, AIST, Waseda University, Tokyo, Japan

    Chie Motono

  9. Institute of Biochemistry and Molecular Biology, Faculty of Medicine, University of Bonn, Bonn, Germany

    Thomas Becker

  10. CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Nikolaus Pfanner & Nils Wiedemann

  11. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Nikolaus Pfanner & Nils Wiedemann

  12. Institute for Protein Dynamics, Kyoto Sangyo University, Kyoto, Japan

    Toshiya Endo

Authors
  1. Hironori Takeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jon V. Busto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Caroline Lindau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Akihisa Tsutsumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kentaro Tomii
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kenichiro Imai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yu Yamamori
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takatsugu Hirokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chie Motono
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Iniyan Ganesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Lena-Sophie Wenz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Thomas Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Masahide Kikkawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Nikolaus Pfanner
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Nils Wiedemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Toshiya Endo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.E. designed the research and wrote the paper. H.T. performed most of the experiments and wrote the paper. J.V.B., C.L., I.G. and L.-S.W. characterized yeast mutants and studied the organization of the SAM complexes and their interaction with precursor proteins. A.T. performed the cryo-EM measurements and data processing, including single-particle analyses, with supervision from M.K. Y.Y. performed the molecular dynamics simulation and molecular dynamics-assisted model building, with supervision from K.T. N.P., N.W. and T.B. designed and wrote part of the paper. K.I., T.H. and C.M. performed the model building and calculation of the subunit interaction energy. All authors contributed to the analysis and discussion of the results of the experiments.

Corresponding authors

Correspondence to Nikolaus Pfanner, Nils Wiedemann or Toshiya Endo.

Ethics declarations

Competing interests

The authors declare no competing interests

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Radhakrishnan Mahalakshmi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Analysis and purification of the SAMTom40-early intermediate.

a, Wild-type (WT) mitochondria were incubated with radiolabeled WT Tom40 and Tom40G354A mutant precursor proteins (pulse), re-isolated and incubated to follow the assembly (chase) of the precursor proteins by blue native-PAGE and radioimaging. Lane 8 shows a higher contrast image of lane 7 to visualize the assembly into the mature TOM complex. b, Digitonin lysed mitochondria isolated from WT and Tom40G354A strains grown at 23 °C in YPG were analyzed by blue native-PAGE and immunoblotting against Tom22. c, Radiolabeled wild-type (WT) Tom40 and Tom40G354A precursor proteins were imported into isolated WT mitochondria and those with Sam50 lacking the POTRA domain (ΔPOTRA), Tom5 (Δ5), Tom6 (Δ6), Tom7 (Δ7), or Sam37 (Δ37) for 30 min at 25 °C, and solubilized complexes were analyzed by blue-native PAGE and radioimaging. TOM, mature TOM complex; SAM-Tom40, SAMTom40-early and/or SAMTom40-folded, SAM-Tom40-5-6, SAMTom40-5-6, and Tom40-5-6-7, the assembly intermediate containing Tom40, Tom5, Tom6, and Tom7. d, [35S]Tom40G354A precursor was imported into wild-type (WT) and sam37Δ mitochondria for the indicated periods. Where indicated, recombinant Sam37 was imported into sam37Δ mitochondria before [35S]Tom40G354A was imported. The samples were lysed with digitonin and analyzed by native electrophoresis and radioimaging. e, The elution profile of gel filtration of the purified SAMTom40-early intermediate in 0.02% GDN. f, g, SDS-PAGE (f) and blue-native PAGE (g) gels of the SAMTom40-early intermediate stained with Coomassie Brilliant Blue (CBB). Data are representative of two (panels a-c), four (panel d), or five times (f, g) independent experiments.

Source data

Extended Data Fig. 2. Cryo-EM structural determination of the SAMTom40-early intermediate.

a, Representative cryo-EM micrograph image of the SAMTom40-early intermediate. The image is a representative of over thousand images. b, Schematic workflows of the steps followed in data processing leading to the final structures of the SAMTom40-early intermediate. c, Cryo-EM maps of the SAMTom40-early intermediate for different classes (b) filtered by different local resolutions (6σ and 9σ). Local resolution filtered maps at a lower threshold level (1σ) are overlaid in grey. Particle numbers and overall map resolutions are shown on the left. d, Upper left, local map resolutions of the final structures (class 3), with colors according to the scales. Upper right, FSC curves between the EM density maps and the atomic models of the SAMTom40-early intermediate (class 3). Lower, FSC curves between the EM density maps and the atomic models for the class 2 and 5 structures.

Extended Data Fig. 3. Cryo-EM structural determination of the SAMTom40-early intermediate.

a, The cryo-EM density maps (contoured at 7.0σ) and the atomic models for indicated segments of Sam50 and Tom40 in the SAMTom40-early intermediate. The structures of Sam35 and Sam37 are essentially the same as those reported before17. b–d, Secondary structure diagrams of Tom40 and Sam50, forming a hybrid β-barrel in the SAMTom40-early intermediate (b), Sam35 (c), and Sam37 (d) are shown. The regions invisible or unresolved are indicated in grey.

Extended Data Fig. 4. Structural characterization of the Tom40-early folding intermediate on SAM.

a, Interactions between loop 1 (between β1 and β2) and the β14-17 region of Sam50 in the SAMdimer complex (PDB ID: 7BTW). b, Comparison of the structures of the Tom40 in the SAMTom40-early intermediate (red) and in the mature TOM complex (pink). c, HA-tagged cysteine-free (Cfree) Sam50 mutants with cysteine residues introduced at the indicated positions (β1 and β16) were expressed in yeast cells lacking endogenous Sam50. Mitochondria were isolated and treated with 1 mM CuSO4 (oxidant) or 1 mM DTT (reductant) and proteins were analyzed by non-reducing SDS-PAGE followed by immunoblotting with the indicated antibodies. Ox, oxidized form with disulfide-bond formation; Red, reduced form. d, Mitochondria treated with or without CuSO4 (oxidant) were solubilized with digitonin and mixed without (Mito) or with GST bound to glutathione-Sepharose beads (GST) or GST followed by the Tom40 β-signal peptide fused with GST bound to glutathione-Sepharose beads (GST-β). The GST domain contains the C-terminally attached cleavage site for 3 C protease. Then Sam50 bound to the Tom40 β-signal was eluted from the beads by 3 C protease treatment, and proteins were analyzed by non-reducing SDS-PAGE followed by immunoblotting with the anti-HA antibodies. e, Radiolabeled Por1β1-19,C276 (19 β-strands) and Por1β2-19,C276 (18 β-strands) were imported for 30 min at 25 °C into Sam50Cfree and Sam50C130 mitochondria. Subsequently, samples were oxidized with 4-DPS and analyzed on non-reducing SDS-PAGE and radioimaging. Arrowhead, disulfide-bond of Sam50 and Por1 precursor; circle, porin precursor. f–i, Cysteine-free Tom40 (CF) and the indicated double-cysteine mutants Tom40 mutants associated with the SAM complex and those integrated into the mature TOM complex were isolated by FLAG purification (g, i) and HA purification (f, h), respectively, as in Fig. 3a. Proteins were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. L, load (5%); E, elute (50%). Data are representative of three (panel c–i) independent experiments.

Source data

Extended Data Fig. 5 MD simulation of Sam50 and Tom40 in the SAMTom40-early intermediate.

a, Time-course changes of secondary structure for the N-terminal region of Tom40 in the SamTom40-early intermediate were calculated using DSSP63. One of the five trajectories is shown as an example. The leftmost part shows the secondary structure of the mature Tom40 (PDB ID: 6JNF). The red and blue colors indicate the residue in α-helix and β-sheet, respectively. The results showed that the residues around the β1-segment of Tom40 are stable as α-helix for a long period of time during the simulation. The tendency was similar for the other four trajectories. b, Using the trajectories after 100 ns, the root-mean-square fluctuation (RMSF) and the fractions of secondary structure were calculated for each trajectory. The RMSF of each residue of Tom40 (upper left) and Sam50 (upper right) of the trajectory corresponding to (a) was plotted. The fractions of secondary structure calculated by DSSP for the same trajectory of Tom40 (lower left) and Sam50 (lower right) were also plotted. The lower part of each panel indicates the secondary structure of mature Tom40 (PDB ID: 6JNF) and the secondary structure of the initial structure of Sam50. The red and blue colors indicate the residue in α-helix and β-sheet, respectively. c, Snapshots of the structures of the Sam50Tom40-early intermediate at 0 ns, 300 ns (as an example of one state), and 800 ns (as an example of the other state) for one of the trajectories. During the MD simulation, the initially separated Tom40 β5-7 segments develop to form a β-sheet. d, Comparison of the cryo-EM density maps and the model, for β1 and its flanking regions, derived by MD-assisted fitting. e, Stereo view of the cryo-EM density maps for β1 and the surrounding part of the hybrid barrel of Tom40 and Sam50.

Extended Data Fig. 6 EM density map of the folding intermediates of Tom40G354A,Δ2-92 on the SAM complex.

Cryo-EM density maps of SAM-Tom40(G354A) (upper) and SAM-Tom40(G354A,Δ2-92) (lower) intermediates contoured at 6.0s, 3.0s, 2.5s, and 2.0s above average are shown. Loop 4, not β1, is seen for SAM-Tom40(G354A,D2-92).

Extended Data Fig. 7 Modeling of Tom5 and Tom6 bound to the SAMTom40-early intermediate.

a, Structures of Tom5 and Tom6 bound to the SAMTom40-early intermediate (SAMTom40-5-6) modeled by fitting simulation analysis. b, c, Close-up views of Tom6 (b) and Tom5 (c) bound to the SAMTom40-early intermediate. d, Structure of the TOM complex (PDB ID: 6JNF). e, f, Close-up views of Tom6 (e) and Tom5 (f) in the TOM complex. g, Interaction energy of Tom40 with Tom5 and Tom6 in the TOM complex (shown by bars in black). Gray zones represent the residues of Tom40 that are not modeled in the EM structure of the SAMTom40-early intermediate. Purple and blue bars in the upper part of the panel indicate the residues interacting with Tom5 and Tom6, respectively. The estimated total subunit interaction free energy is −139.1 kcal/mol for the SAMTom40-early complex and −197.6 kcal/mol for the TOM complex.

Extended Data Fig. 8 Role of the α-protrusion of Sam37 in Tom40 folding on the SAM complex.

a, b, Behaviour of the variant precursor Tom40M94L (used in Fig. 5e). Radiolabeled Tom40 constructs were analyzed by SDS-PAGE and radioimaging (a). The variant precursor Tom40M94L (shown in lane 1) prevents the generation of a truncated Tom40 via translation initiation from the internal methionine M94. Radiolabelled wild-type (WT) Tom40 and Tom40M94L precursor proteins were imported into isolated WT mitochondria for the indicated periods (min) at 25 °C, and solubilized complexes were analysed by blue-native PAGE and radioimaging (b). TOM, mature TOM complex; SAM-Tom40, SAMTom40-early and/or SAMTom40-folded, SAM-Tom40-5-6, SAMTom40-5-6, and Tom40-5-6-7, the assembly intermediate containing Tom40, Tom5, Tom6, and Tom7. Tom40M94L assembled like WT Tom40. c–i, Analysis of the α-protrusion of Sam37. c, Growth of YPH499 wild type, Sam37Δ186-209 and sam37Δ yeast cells on fermentable (YPD) and non-fermentable (YPG) medium at 19 °C, 24 °C, 30 °C and 36 °C °C (Sam37Δ186-209 is chromosomally expressed and contains a GAG linker at the site of truncation). d, Steady-state protein levels of isolated mitochondria from YPH499 wild type and Sam37Δ186-209 cells, as analyzed by Tris/Tricine gel electrophoresis and immunoblotting. Mitochondria were isolated on non-fermentable (YPG) medium at 23 °C. e, f, Radiolabeled Tom22 (e) and Tom20 (f) precursor proteins were imported into isolated YPH499 wild type, Sam37Δ186-209, and sam37Δ mitochondria for the indicated periods, and the digitonin-solubilized complexes were analyzed by blue native PAGE and autoradiography. TOM, the TOM complex. g–i, Radiolabeled Tom5, Tom6, and Tom7 precursor proteins were imported into isolated YPH499 wild type and Sam37Δ186-209 mitochondria for the indicated periods at 25 °C, and the digitonin-solubilized complexes were analyzed by blue native PAGE and radioimaging. TOM, the TOM complex; TOM40-5-6-7, the assembly intermediate consisting of Tom40, Tom5, Tom6, and Tom7. Data are representative of two (panels a, b, d, g–i) or three (panels c, e, f) independent experiments.

Source data

Extended Data Fig. 9 Mitochondrial β-signal versus bacterial β-signal interactions with the β-barrel assembly machineries.

a, Alignment of mitochondrial and bacterial β-barrel protein sequences (left) containing the C-terminal β-signal strand (green lettering)19,26,33,36,64. The β-signal consensus of mitochondria and bacteria is highlighted19. Alignment of Sam50 and BamA sequences (right) containing β1 (underlined in blue). The consensus with the highly conserved glycine on the C-terminal side of β1 allows a direct comparison between bacteria and mitochondria. C.c., Caulobacter crescentus; C.g., Candida glabrata; D.m., Drosophila melanogaster; E.c., Escherichia coli; H.s., Homo sapiens; N.c., Neurospora crassa; N.m., Neisseria meningitidis; R.p., Rickettsia prowazekii; S.c. Saccharomyces cerevisiae; V.c., Vibrio cholerae. b, Cartoon representation of the hybrid interaction between the C-terminal β-signal (green lettering)-containing strand according to structural or crosslinking data (shaded green) of the β-barrel substrates with β1 of Sam50 or BamA, respectively (shaded blue). For the bacterial BAM machinery, the β-signal-strand of several substrates was found to associate with its N-terminl part with BamA β1 to form an asymmetric intermediate (register shift) creating a C-terminal overhang (shaded red) of the β-signal of the substrate (lower panel)33,35. It was proposed that β1 of these substrates can begin to form hydrogen bonds with the overhang of its own C-terminal β-signal-strand to start the barrel closure followed by sequential disruption of the hydrogen bonds between the β-signal-strand and BamA33. In contrast for the mitochondrial SAM machinery, the β-signal-strands of the Tom40 and Por1 substrates are specifically associated with Sam50 β1 without a register shift (upper panel)23, as also observed for the bacterial substrate EspP34,36. Thus, all analyzed mitochondrial β-signals (and one bacterial substrate) associate with Sam50/BamA β1 without a C-terminal overhang, implying a direct β-strand exchange mechanism for barrel closure and release23. c, Interactions between BamA (PDB ID: 6V05) and its substrate (second BamA) with its close-up view with C-terminal overhang. Hydrophilic and hydrophobic interactions are shown with brown dotted lines and black broken lines, respectively. Residues facing the barrel lumen and lipid phase are shown in black and gray, respectively.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Peer Review file

Supplementary Video 1

Five trajectories of molecular dynamics simulation of Tom40 in the SAMTom40-early intermediate. The five 1-μs trajectories of molecular dynamics simulations of the SAMTom40-early intermediate were packed into the continuous animation. The N-terminal region (residues 76–100) of Tom40 is highlighted in red. To fix the point of view, each trajectory was superimposed on the top view of the complex. Animation of the trajectories was done using VMD (Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graphics Modell. 14, 33–38 (1996)).

Source data

Source Data Fig. 1

Uncropped gels and plates.

Source Data Fig. 2

Uncropped gels and plates.

Source Data Fig. 3

Uncropped blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Uncropped blots.

Source Data Fig. 5

Uncropped gels.

Source Data Fig. 6

Uncropped gels.

Source Data Extended Data Fig. 1

Uncropped gels.

Source Data Extended Data Fig. 4

Uncropped gels and blots.

Source Data Extended Data Fig. 8

Uncropped gels, blots and plates.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Takeda, H., Busto, J.V., Lindau, C. et al. A multipoint guidance mechanism for β-barrel folding on the SAM complex. Nat Struct Mol Biol 30, 176–187 (2023). https://doi.org/10.1038/s41594-022-00897-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-022-00897-2

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing