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Cell Chem Biol DOI:10.1016/j.chembiol.2017.02.002

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

Publication TypeJournal Article
Year of Publication2017
AuthorsHan, S, Udeshi, ND, Deerinck, TJ, Svinkina, T, Ellisman, MH, Carr, SA, Ting, AY
JournalCell Chem Biol
Volume24
Issue3
Pages404-414
Date Published2017 Mar 16
ISSN2451-9456
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.

DOI10.1016/j.chembiol.2017.02.002
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/28238724?dopt=Abstract

Alternate JournalCell Chem Biol
PubMed ID28238724
Grant ListR01 CA186568 / CA / NCI NIH HHS / United States