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Isolation of Contact Sites Between Inner and Outer Mitochondrial Membranes

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1567))

Abstract

Mitochondria are essential organelles of all eukaryotic cells. They perform a plethora of important metabolic functions and have a highly complex architecture that differs drastically between different cells and tissues. Mitochondria are delimited from the cytosol by the mitochondrial envelope that consists of the outer membrane and the inner membrane. The inner membrane is subdivided into the inner boundary membrane that runs parallel to the outer membrane and the crista membrane. Both sections of the inner membrane are linked by crista junctions. A further important architectural element of mitochondria are the contact sites between outer membrane and inner membrane. These sites were observed a long time ago by classical electron microscopy, but their molecular structure was identified only recently when it was recognized that proteins of crista junctions and proteins of the outer membrane are responsible for these strong contacts. Mitochondrial function is severely affected when contact sites are disturbed. This underlines the notion that mitochondrial architecture and function are intimately connected. In the following a method is described to generate and to isolate membrane vesicles from isolated yeast mitochondria that contain these contact sites.

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References

  1. Hackenbrock CR (1968) Ultrastructural bases for metabolically linked mechanical activity in mitochondria. II. Electron transport-linked ultrastructural transformations in mitochondria. J Cell Biol 37(2):345–369

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hackenbrock CR (1966) Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria. J Cell Biol 30(2):269–297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Perkins GA et al (1997) Electron tomography of large, multicomponent biological structures. J Struct Biol 120(3):219–227

    Article  CAS  PubMed  Google Scholar 

  4. Perkins GA et al (1998) Electron tomography of mitochondria from brown adipocytes reveals crista junctions. J Bioenerg Biomembr 30(5):431–442

    Article  CAS  PubMed  Google Scholar 

  5. Harner M et al (2011) The mitochondrial contact site complex, a determinant of mitochondrial architecture. EMBO J 30(21):4356–4370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Korner C et al (2012) The C-terminal domain of Fcj1 is required for formation of crista junctions and interacts with the TOB/SAM complex in mitochondria. Mol Biol Cell 23(11):2143–2155

    Article  PubMed  PubMed Central  Google Scholar 

  7. Xie J et al (2007) The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11. FEBS Lett 581(18):3545–3549

    Article  CAS  PubMed  Google Scholar 

  8. Zerbes RM et al (2012) Role of MINOS in mitochondrial membrane architecture: cristae morphology and outer membrane interactions differentially depend on mitofilin domains. J Mol Biol 422(2):183–191

    Article  CAS  PubMed  Google Scholar 

  9. Darshi M et al (2011) ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function. J Biol Chem 286(4):2918–2932

    Article  CAS  PubMed  Google Scholar 

  10. Bohnert M et al (2012) Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane. Mol Biol Cell 23(20):3948–3956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Schleyer M, Neupert W (1985) Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell 43(1):339–350

    Article  CAS  PubMed  Google Scholar 

  12. Schwaiger M, Herzog V, Neupert W (1987) Characterization of translocation contact sites involved in the import of mitochondrial proteins. J Cell Biol 105(1):235–246

    Article  CAS  PubMed  Google Scholar 

  13. Brdiczka DG, Zorov DB, Sheu SS (2006) Mitochondrial contact sites: their role in energy metabolism and apoptosis. Biochim Biophys Acta 1762(2):148–163

    Article  CAS  PubMed  Google Scholar 

  14. Bucheler K, Adams V, Brdiczka D (1991) Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria. Biochim Biophys Acta 1056(3):233–242

    Article  CAS  PubMed  Google Scholar 

  15. Speer O et al (2005) Octameric mitochondrial creatine kinase induces and stabilizes contact sites between the inner and outer membrane. Biochem J 385(Pt 2):445–450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Chacinska A et al (2003) Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex. EMBO J 22(20):5370–5381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Rojo M et al (1991) Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes. J Biol Chem 266:20290–29295

    CAS  PubMed  Google Scholar 

  18. Neupert W (2015) A perspective on transport of proteins into mitochondria: a myriad of open questions. J Mol Biol 427(6 Pt A):1135–1158

    Article  CAS  PubMed  Google Scholar 

  19. Waegemann K et al (2015) Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria. J Mol Biol 427(5):1075–1084

    Article  CAS  PubMed  Google Scholar 

  20. Daum G (1985) Lipids of mitochondria. Biochim Biophys Acta 822(1):1–42

    Article  CAS  PubMed  Google Scholar 

  21. Hovius R et al (1990) Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim Biophys Acta 1021:217–226

    Article  CAS  PubMed  Google Scholar 

  22. Hallermayer G, Neupert W (1974) Lipid composition of mitochondrial outer and inner membranes of Neurospora crassa. Hoppe Seylers Z Physiol Chem 355(3):279–288

    Article  CAS  PubMed  Google Scholar 

  23. Werner S, Neupert W (1972) Functional and biogenetical heterogeneity of the inner membrane of rat-liver mitochondria. Eur J Biochem 25(2):379–396

    Article  CAS  PubMed  Google Scholar 

  24. Riezman H et al (1983) The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles. EMBO J 2(7):1105–1111

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Ohlendieck K et al (1986) Enrichment and biochemical characterization of boundary membrane contact sites from rat-liver mitochondria. Biochim Biophys Acta 860(3):672–689

    Article  CAS  PubMed  Google Scholar 

  26. Sottocasa GL et al (1967) An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol 32(2):415–438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pon L et al (1989) Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins. J Cell Biol 109(6 Pt 1):2603–2616

    Article  CAS  PubMed  Google Scholar 

  28. Donzeau M et al (2000) Tim23 links the inner and outer mitochondrial membranes. Cell 101(4):401–412

    Article  CAS  PubMed  Google Scholar 

  29. Popov-Celeketic D et al (2008) Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria. EMBO J 27(10):1469–1480

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Harner M, Neupert W, Deponte M (2011) Lateral release of proteins from the TOM complex into the outer membrane of mitochondria. EMBO J 30(16):3232–3241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pfanner N et al (2014) Uniform nomenclature for the mitochondrial contact site and cristae organizing system. J Cell Biol 204(7):1083–1086

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to Max Harner .

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Harner, M. (2017). Isolation of Contact Sites Between Inner and Outer Mitochondrial Membranes. In: Mokranjac, D., Perocchi, F. (eds) Mitochondria. Methods in Molecular Biology, vol 1567. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6824-4_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6824-4_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6822-0

  • Online ISBN: 978-1-4939-6824-4

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