title: Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography.
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.
authors.affiliation: Department of Structural Biology, Max-Planck-Institute of Biophysics, 60438, Frankfurt am Main, Germany.|Living Systems Institute, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: Vicki A M
authors.initials: VA
authors.lastname: Gold
authors.affiliation: Department of Structural Biology, Max-Planck-Institute of Biophysics, 60438, Frankfurt am Main, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Brandt
authors.affiliation: Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR8226, 75005, Paris, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Cavellini
authors.affiliation: Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR8226, 75005, Paris, France.
authors.forename: Mickael M
authors.initials: MM
authors.lastname: Cohen
authors.affiliation: Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104, Freiburg, Germany.|Laboratoire de Microbiologie et Génétique Moléculaire (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS,UPS, 31062, Toulouse, France.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Ieva
authors.affiliation: Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104, Freiburg, Germany. Martin.van-der-laan@uks.eu.|BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104, Freiburg, Germany. Martin.van-der-laan@uks.eu.|Medical Biochemistry and Molecular Biology, Saarland University, 66421, Homburg, Germany. Martin.van-der-laan@uks.eu.
authors.forename: Martin
authors.initials: M
authors.lastname: van der Laan
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000379:methods; D055032:Electron Microscope Tomography / Q000379:methods; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D046912:Multiprotein Complexes / Q000378:metabolism; D009154:Mutation; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012984:Software; D013223:Statistics as Topic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D046912:Multiprotein Complexes; D013762:Tetrahydrofolate Dehydrogenase
keywords: Electron cryo-tomography; Membrane proteins; Mitochondria; Presequence translocase; Protein translocation; Quantum dots; TOM complex