title: Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells.
abstract: A recurring challenge in cell biology is to define the molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.
authors.affiliation: Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Shuo
authors.initials: S
authors.lastname: Han
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: Namrata D
authors.initials: ND
authors.lastname: Udeshi
authors.affiliation: National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: Tanya
authors.initials: T
authors.lastname: Svinkina
authors.affiliation: National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
authors.affiliation: Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, CA 94305, USA. Electronic address: ayting@stanford.edu.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D055162:Biocatalysis; D019921:Biotinylation; D004272:DNA, Mitochondrial / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; C000622952:FASTKD1 protein, human; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D009195:Peroxidase; D042967:Electron Transport Complex I; C114961:MT-ND3 protein, human
keywords: APEX; APEX2; FASTKD1; mapping; mass spectrometry; mitochondria; nucleoid; oxphos; peroxidase; proteomics