SpringerLink
Log in

Modular Assembly of Mitochondrial β-Barrel Proteins

  • Protocol
  • First Online:
Transmembrane β-Barrel Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2778))

  • 426 Accesses

Abstract

Mitochondrial β-barrel proteins fulfill crucial roles in the biogenesis and function of the cell organelle. They mediate the import and membrane insertion of proteins and transport of small metabolites and ions. All β-barrel proteins are made as precursors on cytosolic ribosomes and are imported into mitochondria. The β-barrel proteins fold and assemble with partner proteins in the outer membrane. The in vitro import of radiolabelled proteins into isolated mitochondria is a powerful tool to investigate the import of β-barrel proteins, the folding of the β-barrel proteins, and their assembly into protein complexes. Altogether, the in vitro import assay is a versatile and crucial assay to analyze the mechanisms of the biogenesis of mitochondrial β-barrel proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
EUR 44.95
Price includes VAT (Germany)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR 169.99
Price includes VAT (Germany)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
EUR 213.99
Price includes VAT (Germany)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Archibald JM (2015) Endosymbiosis and eukaryotic cell evolution. Curr Biol 25:911–921

    Article  Google Scholar 

  2. Zimorski V, Ku C, Martin WF, Gould SB (2014) Endosymbiotic theory for organelle origins. Curr Opin Microbiol 22:38–48

    Article  CAS  PubMed  Google Scholar 

  3. Spinelli JB, Haigis MC (2018) The multifaceted contributions of mitochondria to cellular metabolism. Nat Cell Biol 20:745–754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Pfanner N, Warscheid B, Wiedemann N (2019) Mitochondrial proteins: from biogenesis to functional networks. Nat Rev Mol Cell Biol 20:267–284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Morgenstern M, Stiller SB, Lübbert P, Peikert CD, Dannenmaier S, Drepper F et al (2017) Definition of a high-confidence mitochondrial proteome at quantitative scale. Cell Rep 19:2836–2852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Morgenstern M, Peikert CD, Lübbert P, Suppanz I, Klemm C, Alka A et al (2021) Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context. Cell Metab 33:2464–2483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rath S, Sharma R, Gupta R, Ast T, Chan C, Durham TJ et al (2021) MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res 49:1541–1547

    Article  Google Scholar 

  8. Hansen KG, Herrmann JM (2019) Transport of proteins into mitochondria. Protein J 38:330–342

    Article  CAS  PubMed  Google Scholar 

  9. Araiso Y, Imai K, Endo T (2022) Role of the TOM complex in protein import into mitochondria: structural views. Annu Rev Biochem 91:679–703

    Article  CAS  PubMed  Google Scholar 

  10. Kutik S, Stojanovski D, Becker L, Becker T, Meinecke M, Krüger V et al (2008) Dissecting membrane insertion of mitochondrial β-barrel proteins. Cell 132:1011–1024

    Article  CAS  PubMed  Google Scholar 

  11. Zeth K (2010) Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology. Biochim Biophys Acta 1797:1292–1299

    Article  CAS  PubMed  Google Scholar 

  12. Webb CT, Heinz E, Lithgow T (2012) Evolution of the β-barrel assembly machinery. Trends Microbiol 20:612–620

    Article  CAS  PubMed  Google Scholar 

  13. Hill K, Model K, Ryan MT, Dietmeier K, Martin F, Wagner R et al (1998) Tom40 forms the hydrophilic channel of the mitochondrial import pore for precursor proteins. Nature 395:516–521

    Article  CAS  PubMed  Google Scholar 

  14. Becker L, Bannwarth M, Meisinger C, Hill K, Model K, Krimmer T et al (2005) Precursor protein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore. J Mol Biol 353:1011–1020

    Article  CAS  PubMed  Google Scholar 

  15. Suzuki H, Kadowaki T, Maeda M, Sasaki H, Nabekura J, Sakaguchi M et al (2004) Membrane-embedded C-terminal segment of rat mitochondrial TOM40 constitutes protein-conducting pore with enriched β-structure. J Biol Chem 279:50619–50629

    Article  CAS  PubMed  Google Scholar 

  16. Shiota T, Imai K, Qiu J, Hewitt VL, Tan K, Shen HH et al (2015) Molecular architecture of the active mitochondrial protein gate. Science 349:1544–1548

    Article  CAS  PubMed  Google Scholar 

  17. Bausewein T, Mills DJ, Langer JD, Nitschke B, Nussberger S, Kühlbrandt W (2017) Cryo-EM structure of the TOM core complex from Neurospora crassa. Cell 170:693–700

    Article  CAS  PubMed  Google Scholar 

  18. Araiso Y, Tsutsumi A, Qiu J, Imai K, Shiota T, Song J et al (2019) Structure of the mitochondrial import gate reveals distinct precursor protein paths. Nature 575:395–401

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gentle I, Gabriel K, Beech P, Waller R, Lithgow T (2004) The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria. J Cell Biol 164:19–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Paschen SA, Waizenegger T, Stan T, Preuss M, Cyrklaff M, Hell K et al (2003) Evolutionary conservation of biogenesis of β-barrel membrane proteins. Nature 426:862–866

    Article  CAS  PubMed  Google Scholar 

  22. Kozjak V, Wiedemann N, Milenkovic D, Lohaus C, Meyer HE, Guiard B et al (2003) An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane. J Biol Chem 278:48520–48523

    Article  CAS  PubMed  Google Scholar 

  23. Höhr AIC, Lindau C, Wirth C, Qiu J, Stroud DA, Kutik S et al (2018) Membrane protein insertion through a mitochondrial β-barrel gate. Science 359:eaah6834

    Article  PubMed  PubMed Central  Google Scholar 

  24. Takeda H, Tsutsumi A, Nishizawa T, Lindau C, Busto JV, Wenz LS et al (2021) Mitochondrial sorting and assembly machinery operates by β-barrel switching. Nature 590:163–169

    Article  CAS  PubMed  Google Scholar 

  25. Takeda H, Busto JV, Lindau C, Tsutsumi A, Tomii K, Imai K et al (2023) A multipoint guidance mechanism for β-barrel folding on the SAM complex. Nat Struct Mol Biol 30:176–187

    Article  CAS  PubMed  Google Scholar 

  26. Colombini M (2012) Mitochondrial outer membrane channels. Chem Rev 112:6373–6387

    Article  CAS  PubMed  Google Scholar 

  27. Campo ML, Peixoto PM, Martínez-Caballero S (2017) Revisiting trends on mitochondrial mega-channels for the import of proteins and nucleic acids. J Bioenerg Biomembr 49:75–99

    Article  CAS  PubMed  Google Scholar 

  28. Becker T, Wagner R (2018) Mitochondrial outer membrane channels: emerging diversity in transport processes. BioEssays 40:e1800013

    Article  PubMed  Google Scholar 

  29. Meisinger C, Rissler M, Chacinska A, Szklarz LK, Milenkovic D, Kozjak V et al (2004) The mitochondrial morphology protein Mdm10 functions in assembly of the precursor protein translocase of the outer membrane. Dev Cell 7:61–71

    Article  CAS  PubMed  Google Scholar 

  30. Meisinger C, Pfannschmidt S, Rissler M, Milenkovic D, Becker T, Stjanovski D et al (2007) The morphology proteins Mdm12/Mmm1 function in the major β-barrel assembly pathway of mitochondria. EMBO J 26:2229–2239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kornmann B, Currie E, Collins SR, Schuldiner M, Nunnari J, Weissman JS et al (2009) An ER-mitochondria tethering complex revealed by a synthetic biology screen. Science 325:477–481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yamano K, Tanaka-Yamano S, Endo T (2010) Tom7 regulates Mdm10-mediated assembly of the mitochondrial import channel protein Tom40. J Biol Chem 285:41222–41231

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Klein A, Israel L, Lackey SW, Nargang FE, Imhof A, Baumeister W et al (2012) Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane. J Cell Biol 199:599–611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ellenrieder L, Opaliński Ł, Becker L, Krüger V, Mirus O, Straub SP et al (2016) Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10. Nat Commun 7:13021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hiller S, Garces RG, Malia TJ, Orekhov VY, Colombini M, Wagner G (2008) Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 321:1206–1210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bayrhuber M, Meins T, Habeck M, Becker S, Giller K, Villinger S et al (2008) Structure of the human voltage-dependent anion channel. Proc Natl Acad Sci U S A 105:15370–15375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ujwal R, Cascio D, Colletier JP, Faham S, Zhang J, Toro L et al (2008) The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating. Proc Natl Acad Sci 105:17742–17747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Flinner N, Ellenrieder L, Stiller SB, Becker T, Schleiff E, Mirus O (2013) Mdm10 is an ancient eukaryotic porin co-occurring with the ERMES complex. Biochim Biophys Acta 1833:3314–3325

    Article  CAS  PubMed  Google Scholar 

  39. Diederichs KA, Ni X, Rollauer SE, Botos I, Tan X, King MS et al (2020) Structural insight into mitochondrial β-barrel outer membrane protein biogenesis. Nat Commun 11:3290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jores T, Lawatscheck J, Beke V, Franz-Wachtel M, Yunoki K, Fitzgerald JC et al (2018) Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial β-barrel proteins. J Cell Biol 217:3091–3108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Jores T, Klinger A, Groß LE, Kawano S, Flinner N, Duchardt-Ferner E et al (2016) Characterization of the targeting signal in mitochondrial β-barrel proteins. Nat Commun 7:12036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wiedemann N, Kozjak V, Chacinska A, Schönfisch B, Rospert S, Ryan MT et al (2003) Machinery for protein sorting and assembly in the mitochondrial outer membrane. Nature 424:565–571

    Article  CAS  PubMed  Google Scholar 

  43. Qiu J, Wenz LS, Zerbes RM, Oeljeklaus S, Bohnert M, Stroud DA et al (2013) Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation. Cell 154:596–608

    Article  CAS  PubMed  Google Scholar 

  44. Wenz LS, Ellenrieder L, Qiu J, Bohnert M, Zufall N, van der Laan M et al (2015) Sam37 is crucial for formation of the mitochondrial TOM-SAM supercomplex, thereby promoting β-barrel biogenesis. J Cell Biol 210:1047–1054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wiedemann N, Truscott KN, Pfannschmidt S, Guiard B, Meisinger C, Pfanner N (2004) Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway. J Biol Chem 279:18188–18194

    Article  CAS  PubMed  Google Scholar 

  46. Hoppins SC, Nargang FE (2004) The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes. J Biol Chem 279:12396–12405

    Article  CAS  PubMed  Google Scholar 

  47. Weinhäupl K, Lindau C, Hessel A, Wang Y, Schütze C, Jores T et al (2018) Structural basis of membrane protein chaperoning through the mitochondrial intermembrane space. Cell 175:1365–1379

    Article  PubMed  PubMed Central  Google Scholar 

  48. Noinaj N, Gumbart JC, Buchanan SK (2017) The β-barrel assembly machinery in motion. Nat Rev Microbiol 15:197–204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang Q, Guan Z, Qi L, Zhuang J, Wang C, Hong S, Yan L et al (2021) Structural insight into the SAM-mediated assembly of the mitochondrial TOM core complex. Science 373:1377–1381

    Article  CAS  PubMed  Google Scholar 

  50. Becker T, Guiard B, Thornton N, Zufall N, Stroud DA, Wiedemann N et al (2010) 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Thornton N, Stroud DA, Milenkovic D, Guard B, Pfanner N, Becker T (2010) Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of α-helical outer membrane proteins. J Mol Biol 396:540–549

    Article  CAS  PubMed  Google Scholar 

  52. Becker T, Wenz LS, Thornton N, Stroud D, Meisinger C, Wiedemann N et al (2011) Biogenesis of mitochondria: dual role of Tom7 in modulating assembly of the precursor protein translocase of the outer membrane. J Mol Biol 405:113–124

    Article  CAS  PubMed  Google Scholar 

  53. Rapaport D, Neupert W (1999) Biogenesis of Tom40, core component of the TOM complex of mitochondria. J Cell Biol 146:321–331

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Stroud DA, Becker T, Qiu J, Stojanovski D, Pfannschmidt S, Wirth C et al (2011) Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex. Mol Biol Cell 22:2823–2833

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Priesnitz C, Pfanner N, Becker T (2020) Studying protein import into mitochondria. Methods Cell Biol 155:45–79

    Article  CAS  PubMed  Google Scholar 

  56. Chen LC, Casadevall A (1999) Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells. Anal Biochem 269:179–188

    Article  CAS  PubMed  Google Scholar 

  57. Wang F, Brown EC, Mak G, Zhuang J, Denic V (2010) A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum. Mol Cell 40:159–171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Fünfschilling U, Rospert S (1999) Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria. Mol Biol Cell 10:3289–3299

    Article  PubMed  PubMed Central  Google Scholar 

  59. Schuldiner M, Metz J, Schmid V, Denic V, Rakwalska M, Schmitt HD et al (2008) The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134:634–645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Pelham HR, Jackson RJ (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem 67:247–256

    Article  CAS  PubMed  Google Scholar 

  61. Meisinger C, Pfanner N, Truscott KN (2006) Isolation of yeast mitochondria. Methods Mol Biol 313:33–39

    CAS  PubMed  Google Scholar 

  62. Mokranjac D, Neupert W (2007) Protein import into isolated mitochondria. Methods Mol Biol 372:277–286

    Article  CAS  PubMed  Google Scholar 

  63. Krimmer T, Rapaport D, Ryan MT, Meisinger C, Kassenbrock CK, Blachly-Dyson E et al (2001) Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex. J Cell Biol 152:289–300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Habib SJ, Waizenegger T, Lech M, Neupert W, Rapaport D (2005) Assembly of the TOB complex of mitochondria. J Biol Chem 280:6434–6440

    Article  CAS  PubMed  Google Scholar 

  65. Humphries AD, Streinmann IC, Stojanovski D, Johnston AJ, Yano M, Hoogenraad NJ et al (2005) Dissection of the mitochondrial import and assembly pathway of human Tom40. J Biol Chem 280:11535–11543

    Article  CAS  PubMed  Google Scholar 

  66. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231

    Article  PubMed  Google Scholar 

  67. Schulte U, den Brave F, Haupt A, Gupta A, Song J, Müller CS et al (2023) Mitochondrial complexome reveals quality-control pathways of protein import. Nature 614:153–159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Schägger H, Pfeiffer K (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19:1777–1783

    Article  PubMed  PubMed Central  Google Scholar 

  69. Dekker PJ, Martin F, Maarse AC, Bömer U, Müller H, Guiard B et al (1997) The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested precursor proteins in the absence of matrix Hsp70-Tim44. EMBO J 16:5408–5419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  71. Fujiki Y, Hubbard AL, Fowler S, Lazarow PB (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102

    Article  CAS  PubMed  Google Scholar 

  72. Doan KN, Grevel A, Mårtensson CU, Ellenrieder L, Thornton N, Wenz LS et al (2020) The mitochondrial import complex MIM functions as main translocase for α-helical outer membrane proteins. Cell Rep 31:107567

    Article  CAS  PubMed  Google Scholar 

  73. Truscott KN, Pfannschmidt S, Guiard B, Meisinger C, Pfanner N (2003) A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria. J Cell Biol 163:707–713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Nicole Zufall for expert technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG), BE4679/2-2 project ID 269424439 (to T.B.) and SFB1218 project ID 269925409 (to T.B.).

Author information

Author notes

  1. Rituparna Bhowmik and Fabian den Brave contributed equally with all other contributors.

Authors and Affiliations

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

    Rituparna Bhowmik, Fabian den Brave & Thomas Becker

Authors
  1. Rituparna Bhowmik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fabian den Brave
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Becker .

Editor information

Editors and Affiliations

  1. Laboratoire de Microbiologie et Génétique Moléculaires, Centre de Biologie Intégrative, CNRS, Université de Toulouse, Toulouse, France

    Raffaele Ieva

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Bhowmik, R., den Brave, F., Becker, T. (2024). Modular Assembly of Mitochondrial β-Barrel Proteins. In: Ieva, R. (eds) Transmembrane β-Barrel Proteins. Methods in Molecular Biology, vol 2778. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3734-0_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3734-0_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3733-3

  • Online ISBN: 978-1-0716-3734-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation