2. Academic Validation
  3. Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

  • Cell Chem Biol. 2017 Mar 16;24(3):404-414. doi: 10.1016/j.chembiol.2017.02.002.
Shuo Han 1,  Namrata D Udeshi 2,  Thomas J Deerinck 3,  Tanya Svinkina 2,  Mark H Ellisman 3,  Steven A Carr 2,  Alice Y Ting 4
Affiliations

Affiliations

  • 1 Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, CA 94305, USA.
  • 2 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
  • 3 National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, CA 92093, USA.
  • 4 Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, CA 94305, USA. Electronic address: [email protected].
PMID: 28238724 DOI: 10.1016/j.chembiol.2017.02.002
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.

Keywords

APEX; APEX2; FASTKD1; mapping; mass spectrometry; mitochondria; nucleoid; oxphos; peroxidase; proteomics.

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