Characterizing the mitochondrial DNA polymerase gamma interactome by BioID identifies Ruvbl2 localizes to the mitochondria Sanduni U. Liyanage a,b , Etienne Coyaud a , Estelle M.N. Laurent a , Rose Hurren a , Neil Maclean a , Stuart R. Wood c,1 , Lawrence Kazak c,2 , Aisha Shamas-Din a , Ian Holt c , Brian Raught a , Aaron Schimmer a,b, ⁎ a Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada b Department of Medical Biophysics, University of Toronto, ON, Canada c Medical Research Council, Mitochondrial Biology Unit, Cambridge, UK abstract article info Article history: Received 23 August 2016 Received in revised form 13 October 2016 Accepted 2 November 2016 Available online 11 November 2016 Human mitochondrial DNA (mtDNA) is replicated by the mitochondrial DNA polymerase gamma (POLG). Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new inter- action partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this pro- tein. Ruvbl2 was detected in mitochondria-enriched fractions in leukemic cells. Additionally, transgenic overex- pression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a po- tential novel mitochondrial isoform of Ruvbl2. © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: POLG Ruvbl2 mtDNA Nucleoid BioID Co-localization 1. Introduction Human mitochondrial DNA is a 16.6 kb genome (mtDNA) which en- codes genes essential for oxidative metabolism (Chan and Copeland, 2009). It is replicated by the nuclear-encoded mitochondrial DNA poly- merase gamma (POLG). Human POLG consists of a C-terminal catalytic polymerase domain and an N-terminal exonuclease domain separated by a linker region. The holoenzyme consists of the primary subunit POLG and a homodimeric form of its accessory subunit POLG2 (Graziewicz et al., 2006). POLG is located within poorly defined multi- protein-DNA complexes termed nucleoids, a crucial hub conducive to packaging and supporting mtDNA maintenance, transcription and translation (Bogenhagen, 2012). We reasoned that a novel proximity-based biotinylation (BioID) established for identification of protein-protein interactions can be used to identify low abundant, transient interactors of POLG. To identify protein-protein interactions with BioID, the protein of interest is fused with a mutant E. coli biotin conjugating enzyme (BirA R118G, or BirA*) that promiscuously biotinylates nearby proteins. Following cell lysis, biotinylated proteins are affinity purified and identified using mass spectrometry (Roux et al., 2012). Currently, the interactome pro- file of POLG is not characterized. Here, we report novel protein-protein interactions of POLG using the BioID method. 2. Materials and methods 2.1. BioID interactome mapping in cell lines Using the Flp-In system (Invitrogen), HEK 293 T-REx Flp-In cells sta- bly expressing POLG- FlagBirA*, negative controls ornithine transcarbamylase (OTC)- FlagBirA*, and Flag-BirA*- were generated and mass spectrometric experiments performed as previously de- scribed (Coyaud et al., 2015). Parameters for high confidence BioID hits were identified with a Protein Prophet algorithm cut-off of 0.9, followed by analysis with the SAINT express algorithm using a cut-off value set to 0.70. (v3.3). For each protein, total spectral counts of 4 sam- ples (2 biological replicates in duplicate) were analyzed using the SAINT algorithm. 24 negative controls (20 Flag-BirA*only and 4 ornithine transcarbamylase (OTC-FlagBirA*)) were collapsed to the top 4 highest spectral counts for each prey. BioID hits were annotated for cellular lo- calization from Uniprot database, functional classification was deter- mined from Gene-ontology using the DAVID bioinformatics resource Mitochondrion 32 (2017) 31–35 Abbreviations: BioID, proximity dependent biotin labelling. ⁎ Corresponding author at: Princess Margaret Cancer Centre, Room 7-417, 610 University Ave, Toronto, ON M5G 2M9, Canada. E-mail address: aaron.schimmer@utoronto.ca (A. Schimmer). 1 Current address: Discuva Limited, The Merrifield Centre, 12 Rosemary Lane, Cambridge, UK. 2 Current address: Dana-Farber Cancer Institute, Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA. http://dx.doi.org/10.1016/j.mito.2016.11.001 1567-7249/© 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Contents lists available at ScienceDirect Mitochondrion journal homepage: www.elsevier.com/locate/mito