Monitoring the Mitochondrial Presequence Import Pathway In Living Mammalian Cells with a New Molecular Biosensor

Most mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytosol, and imported into the organelle. Mitochondrial protein import is therefore vital for the maintenance of mitochondrial function and cell survival. Alterations in this process are suspected to contribute to various diseases, including neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Our understanding of the cytosolic signaling mechanisms and posttranslational modifications regulating the mitochondrial import process is still in its infancy and hampered by the lack of tools for its dynamic assessment in cells. We recently engineered an inducible molecular biosensor for monitoring one of the main mitochondrial import routes, the so-called presequence pathway, using a quantitative luminescence-based readout. Here, we provide basic guidelines for using this probe in common cell types of general use in the scientific community: HEK293T cells, human fibroblasts, and mouse primary neurons. These guidelines can serve as a starting point for the development of more elaborated protocols for the dynamic investigation of the presequence import pathway and its regulation in relevant physiological and pathological conditions.