Abstract
Mitochondrial nucleoids are compact nucleoprotein complexes, in which mtDNA is located, replicated, and transcribed. Several proteomic approaches have been previously employed to identify nucleoid proteins; however, a consensus list of nucleoid-associated proteins has not been generated. Here we describe a proximity-biotinylation assay, BioID, which allows identification of proximity interactors of mitochondrial nucleoid proteins. It uses a promiscuous biotin ligase fused to a protein of interest which covalently attaches biotin to lysine residues of its proximal neighbors. Biotinylated proteins can be further enriched by a biotin-affinity purification and identified by mass-spectrometry. BioID can identify transient and weak interactions and can be used to identify changes in the interactions upon different cellular treatments, for different protein isoforms or for pathogenic variants.