MitoLuc: A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria

All but a few mitochondrial proteins are translated into the cytosol and imported in via complicated and varied pathways. These processes occur over short time frames and, as such, are difficult to monitor with classical approaches such as Western blotting or autoradiography that require sample collection at discrete time points. The development of an assay based on a split version of the small luciferase-Mitoluc-has allowed us to monitor the import of proteins into mitochondria in high resolution and real time (Pereira et al., J Mol Biol 431:1689-1699, 2019). Luminescence measurements are acquired using a plate reader in the order of seconds. This allows scores of experiments to be conducted in parallel in a single multi-well plate and permits kinetic analysis yielding information about import mechanisms (Ford et al., Elife 11:e75426, 2022).