title: Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells.
abstract: The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.
authors.affiliation: Institute of Molecular Cell Biology, School of Biology, University of Münster, D-48149 Münster, Germany.|Mitochondrial Dynamics Group, School of Biology, University of Osnabrueck, D-49076 Osnabrueck, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Rieger
authors.affiliation: School of Physics, University of Osnabrueck, D-49069 Osnabrueck, Germany.
authors.forename: Daria N
authors.initials: DN
authors.lastname: Shalaeva
authors.affiliation: Institute of Molecular Cell Biology, School of Biology, University of Münster, D-48149 Münster, Germany.|Mitochondrial Dynamics Group, School of Biology, University of Osnabrueck, D-49076 Osnabrueck, Germany.
authors.forename: Anna-Carina
authors.initials: AC
authors.lastname: Söhnel
authors.affiliation: Mitochondrial Dynamics Group, School of Biology, University of Osnabrueck, D-49076 Osnabrueck, Germany.
authors.forename: Wladislaw
authors.initials: W
authors.lastname: Kohl
authors.affiliation: Institute of Molecular Cell Biology, School of Biology, University of Münster, D-48149 Münster, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Duwe
authors.affiliation: School of Physics, University of Osnabrueck, D-49069 Osnabrueck, Germany.|School of Bioengineering and Bioinformatics, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.|A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: Armen Y
authors.initials: AY
authors.lastname: Mulkidjanian
authors.affiliation: Institute of Molecular Cell Biology, School of Biology, University of Münster, D-48149 Münster, Germany.|Mitochondrial Dynamics Group, School of Biology, University of Osnabrueck, D-49076 Osnabrueck, Germany.
authors.forename: Karin B
authors.initials: KB
authors.lastname: Busch
mesh_terms: D019069:Cell Respiration; D002470:Cell Survival; D003576:Electron Transport Complex IV / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D003576:Electron Transport Complex IV