Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography
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 wordsElectron cryo-tomography Quantum dots Mitochondria Protein translocation Membrane proteins TOM complex Presequence translocase
We thank Werner Kühlbrandt for discussion and critical reading of the manuscript. This work was supported by the Max Planck Society (V.A.M.G., T.B.), the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 746) (M.v.d.L), the Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS) (M.v.d.L.), the CNRS-INSERM ATIP-Avenir program (M.C., R.I.), the “fondation pour la recherche médicale” (INE 20100518343) (M.C.), the labex DYNAMO (ANR-11-LABX-0011-DYNAMO) (M.C.), a Marie Curie IRG grant to M.C. (No.276912), and an EMBO Long-Term Fellowship to V.A.M.G. (ALTF 1035-2010).
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