Characterization of a new mitophagy reporter uncovers requirement of BNIP3 for hypoxia-induced mitophagy in Drosophila

Background. Mitophagy is the cellular removal of unwanted mitochondria via the lysosome. Given the importance of this process to energy demanding tissues, mitophagy defects have been linked to various metabolic and neurodegenerative diseases. Mitophagy assessment tools are important for evaluating and quantifying mitophagy flux, which are useful in studying mitophagy pathways, mechanisms, and dysfunction. Mitophagy reporters are commonly used reagents to examine endpoint mitophagy flux. Following the generation of a new mitophagy reporter, mitoSRAI (mitochondrial Signal Retaining Autophagy Indicator), we introduced this reporter as a transgene into Drosophila melanogaster (Dm). We hypothesized that mitoSRAI will be capable of measuring mitophagic flux through microscopic visualization of the TOLLES:YPet fluorescence ratios, and biochemically through the relative persistence of TOLLES proteins in the lysosomes following YPet degradation. Results. We found that when we express the mitoSRAI reporter in the Dm larval muscle wall and examine mitoSRAI flux by inducing mitophagy via hypoxia, we observe a significant increase in TOLLES only fluorescent signals and bands by confocal imaging and western blotting respectively. Complementarily, the readout of mitoSRAI is sensitive to conditions of mitophagy inhibition under hypoxia. To validate our results, we compared mitoSRAI to a similarly constructed reporter, matrix-QC, and found that mitoSRAI is less responsive to neuronal and fat body mitophagy flux manipulations. Conclusion. Overall, our work characterizes the strengths and weaknesses of the application of the mitoSRAI reporter in Dm. We demonstrate with the mitoSRAI reporter that BNIP3 is an important mediator for hypoxia-induced mitophagy in Dm.