The NanoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import within Intact Mammalian Cells

Only a few proteins (13 in humans) are encoded by the mammalian mitochondrial genome. Therefore, the other mitochondrial resident proteins (>1000) must be recruited via specialised import pathways. Protein import is critical for mitochondrial biogenesis and bioenergetic function and health; loss of function has been implicated with a wide range of pathologies. Despite this, our understanding of the kinetic and dynamics of import is somewhat limited, particularly within mammalian cells. Here, we report an adaptation of an assay system, established previously to monitor mitochondrial import into isolated yeast mitochondria, to quantitatively monitor mitochondrial import inside mammalian cells. The reporting is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a recombinant precursor protein destined for import. Following import successively through the TOM complex of the outer membrane and the TIM23 complex of the inner membrane, the complementary fragments combine to form an active luciferase. The resultant luminescent signal provides a sensitive, accurate, free of noise and continuous measure of protein import, enabling mathematical model fitting to identify and understand the steps that make up import. This advance allows detailed mechanistic examination of the transport process in live cells. In addition, the assay will enable characterisation of the protein import when the machinery is challenged; for example, in situations associated with disease. Moreover, the assay is compatible with high throughput for large data set collection and kinetic modelling, as well as for drug screening and characterisation. Our set-up also has the potential to be adapted for the analysis of alternative transport systems and different cell types, and even for multicellular model organisms.