Mitochondria pp 315-336

Part of the Methods in Molecular Biology book series (MIMB, volume 1567) | Cite as

Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography

  • Vicki A. M. Gold
  • Tobias Brandt
  • Laetitia Cavellini
  • Mickael M. Cohen
  • Raffaele Ieva
  • Martin van der Laan
Protocol

Abstract

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.

Key words

Electron cryo-tomography Quantum dots Mitochondria Protein translocation Membrane proteins TOM complex Presequence translocase 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Vicki A. M. Gold
    • 1
    • 7
  • Tobias Brandt
    • 1
  • Laetitia Cavellini
    • 2
  • Mickael M. Cohen
    • 2
  • Raffaele Ieva
    • 3
    • 4
  • Martin van der Laan
    • 3
    • 5
    • 6
  1. 1.Department of Structural BiologyMax-Planck-Institute of BiophysicsFrankfurt am MainGermany
  2. 2.Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-ChimiqueSorbonne Universités, UPMC Univ Paris 06, CNRS, UMR8226ParisFrance
  3. 3.Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of MedicineUniversity of FreiburgFreiburgGermany
  4. 4.Laboratoire de Microbiologie et Génétique Moléculaire (LMGM), Centre de Biologie Intégrative (CBI)Université de Toulouse, CNRS,UPSToulouseFrance
  5. 5.BIOSS Centre for Biological Signalling StudiesUniversity of FreiburgFreiburgGermany
  6. 6.Medical Biochemistry and Molecular BiologySaarland UniversityHomburgGermany
  7. 7.Living Systems InstituteUniversity of ExeterExeterUK

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