Abstract
Mitochondrial protein import is critical for organelle biogenesis, bioenergetic function, and health. The mechanism of which is poorly understood, particularly of the mammalian system. To address this problem we have established an assay to quantitatively monitor mitochondrial import inside mammalian cells. The reporter 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 purified recombinant precursor protein destined for import. Following import the complementary fragments combine to form an active luciferase-providing a sensitive, accurate and continuous measure of protein import. This advance allows detailed mechanistic examination of the transport process in live cells, including the analysis of import breakdown associated with disease, and high-throughput drug screening. Furthermore, the set-up has the potential to be adapted for the analysis of alternative protein transport systems within different cell types, and multicellular model organisms.
Keywords:
in-cell assay; mitochondria; mitoluc; nanoluc; protein import; split luciferase.