BRET-Based Mitochondrial Subcompartment Localization Biosensors Reveal the Impact of Pesticides and Food Additives on Mitochondrial Protein Localization.

The last decade has witnessed a marked increase in interest in mitochondria, whose dysfunction leads to the development of multiple diseases. Mitochondria are unique as they are highly compartmentalized organelles that are composed of two closely apposed membranes. Here, we developed a series of bioluminescence resonance energy transfer (BRET)-based localization sensors that accurately report the localization and orientation of mitochondrial proteins (TOM20, TOM22, VDAC1, MICU1, ATP5F1C, OTC, and SIRT3) within their respective mitochondrial compartments. These biosensors also dynamically detect stressor-induced translocation of cytosolic proteins, such as Drp1 and BAX, to mitochondria. Screening of a series of endocrine disruptors with our BRET sensors uncovered deleterious effects of paraquat, di-tert-butyl-4-methylphenol, and 4-hydroxynonenal on mitochondrial protein localization. Altogether, the localization sensors provide noninvasive tools to monitor mitochondrial protein localization in a time-resolved manner, with nanometer-scale resolution, in intact cells exposed to diverse cellular stressors.