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      The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes*

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          Abstract

          Mitochondria exert an immense amount of cytophysiological functions, but the structural basis of most of these processes is still poorly understood. Here we use cross-linking mass spectrometry to probe the organization of proteins in native mouse heart mitochondria. Our approach provides the largest survey of mitochondrial protein interactions reported so far. In total, we identify 3,322 unique residue-to-residue contacts involving half of the mitochondrial proteome detected by bottom-up proteomics. The obtained mitochondrial protein interactome gives insights in the architecture and submitochondrial localization of defined protein assemblies, and reveals the mitochondrial localization of four proteins not yet included in the MitoCarta database. As one of the highlights, we show that the oxidative phosphorylation complexes I-V exist in close spatial proximity, providing direct evidence for supercomplex assembly in intact mitochondria. The specificity of these contacts is demonstrated by comparative analysis of mitochondria after high salt treatment, which disrupts the native supercomplexes and substantially changes the mitochondrial interactome.

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          Most cited references 69

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          CORUM: the comprehensive resource of mammalian protein complexes—2009

          CORUM is a database that provides a manually curated repository of experimentally characterized protein complexes from mammalian organisms, mainly human (64%), mouse (16%) and rat (12%). Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The new CORUM 2.0 release encompasses 2837 protein complexes offering the largest and most comprehensive publicly available dataset of mammalian protein complexes. The CORUM dataset is built from 3198 different genes, representing ∼16% of the protein coding genes in humans. Each protein complex is described by a protein complex name, subunit composition, function as well as the literature reference that characterizes the respective protein complex. Recent developments include mapping of functional annotation to Gene Ontology terms as well as cross-references to Entrez Gene identifiers. In addition, a ‘Phylogenetic Conservation’ analysis tool was implemented that analyses the potential occurrence of orthologous protein complex subunits in mammals and other selected groups of organisms. This allows one to predict the occurrence of protein complexes in different phylogenetic groups. CORUM is freely accessible at (http://mips.helmholtz-muenchen.de/genre/proj/corum/index.html).
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            Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.

            Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.
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              Directed evolution of APEX2 for electron microscopy and proteomics

              APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
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                Author and article information

                Journal
                Mol Cell Proteomics
                Mol. Cell Proteomics
                mcprot
                mcprot
                MCP
                Molecular & Cellular Proteomics : MCP
                The American Society for Biochemistry and Molecular Biology
                1535-9476
                1535-9484
                February 2018
                8 December 2017
                8 December 2017
                : 17
                : 2
                : 216-232
                Affiliations
                From the ‡Biomolecular Mass Spectrometry and Proteomics. Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands;
                §Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands;
                ¶Leibniz Institute of Molecular Pharmacology (FMP Berlin), Robert-Rössle-Straβe 10, 13125 Berlin, Germany;
                ‖Laboratory of Cardiac Energetics, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
                Author notes
                ‡‡To whom correspondence should be addressed: balabanr@ 123456nhlbi.nih.gov (RSB), a.j.r.heck@ 123456uu.nl (AJRH).

                ** These authors contributed equally to this work.

                Author contributions: F.L., P.L., B.M.R., R.S.B., and A.J.R.H. designed the research; F.L., P.L., and B.M.R. performed the research; F.L. and B.M.R. contributed new reagents/analytic tools; F.L., P.L., R.S.B., and A.J.R.H. analyzed data; and F.L., P.L., B.M.R., R.S.B., and A.J.R.H. wrote the paper.

                Article
                RA117.000470
                10.1074/mcp.RA117.000470
                5795388
                29222160
                © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

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                Molecular biology

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