Detection and quantitation of ferritinophagy using HaloTag tracing

Iron is an essential element required for critical processes, such as oxygen transport, energy generation, and DNA synthesis. To be incorporated as a cofactor, iron that is stored in the cytosol within ferritin needs to be liberated by ferritinophagy. Ferritinophagy is an autophagic process in which ferritin is targeted to the lysosomes, through its interaction with nuclear receptor coactivator 4 for degradation and release of labile iron. Despite its involvement in neurodegenerative diseases, anemia, cancer, and insulin resistance, a specific and sensitive method to detect ferritinophagy has been lacking. To detect and quantitate ferritinophagic flux, we generated a Halo-tagged ferritin heavy chain 1 (FTH1) construct and took advantage of stabilization of Halo fragment in the presence of its fluorescently labeled ligand. Stably expressed Halo-FTH1 operated identical to its endogenous counterpart. More importantly, using pulse-chase settings, lysosomal accumulation of Halo fragment after induction of ferritinophagy was detected and quantitated by in-gel fluorescence, immunoblotting, and microscopic analyses. Finally, we found that silencing of nuclear receptor coactivator 4 prevented accumulation of tetramethylrhodamine-Halo fragment and degradation of endogenous FTH1 under ferritinophagic conditions, confirming the specificity of our assay. Together, the HaloTag-FTH1 tool we generated can be used to specifically detect and quantitate ferritinophagy in mammalian cells with a fluorescent Halo ligand, and this approach can be instrumental in studies focusing on cellular iron metabolism.