Aaltonen Mari J, Antonicka Hana
Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
Department of Human Genetics, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2023;2615:153-172. doi: 10.1007/978-1-0716-2922-2_12.
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.
线粒体类核是紧密的核蛋白复合体，线粒体DNA位于其中进行复制和转录。此前已采用多种蛋白质组学方法来鉴定类核蛋白；然而，尚未生成一份类核相关蛋白的共识清单。在此，我们描述了一种邻近生物素化分析方法——BioID，它能够鉴定线粒体类核蛋白的邻近相互作用蛋白。该方法利用一种与目标蛋白融合的泛素连接酶，将生物素共价连接到其邻近蛋白的赖氨酸残基上。生物素化的蛋白可通过生物素亲和纯化进一步富集，并通过质谱进行鉴定。BioID能够鉴定瞬时和弱相互作用，可用于鉴定在不同细胞处理条件下、针对不同蛋白质异构体或致病变体时相互作用的变化。
