Mitochondria Protein Import: Methods

Abstract Mitochondria are prominent and essential organelles within eukaryotic cells. The majority of proteins residing within mitochondria are nuclear encoded and consequently synthesised as precursor proteins on cytosolic ribosomes. These precursors must be imported across one or both of the mitochondrial membranes to reach their final destination where they can acquire their functional state. The import proceeds owing to the presence of different pathways formed by sophisticated complexes known as the import complexes. The complexes are located in all mitochondrial compartments including both mitochondrial membranes. Currently, we have knowledge of at least four different pathways that govern the import and sorting of nuclear‐encoded mitochondrial precursors. The elucidation and characterisation of these pathways has been greatly facilitated by the in vitro mitochondrial import assay. The in vitro assay is the main biochemical approach for analysing protein import into mitochondria and is performed by incubating a radiolabelled precursor protein with isolated mitochondria. Key Concepts Most mitochondrial proteins are encoded by nuclear DNA and need to be imported into the organelle following translation. Different pathways exist for the import of proteins into the various mitochondrial subcompartments. In vitro translated precursors can be imported into isolated mitochondria. The final residence of the imported precursor can be analysed using various treatments. In vitro import reactions can be accompanied with monitoring the assembly of the precursor into complexes.