The inositol pyrophosphate 5-InsP₇ regulates mitochondrial polyphosphate synthesis and bioenergetic function

Inorganic polyphosphate (polyP) is a linear polymer of phosphate residues linked by phosphoanhydride bonds. PolyP remains poorly understood in mammals due to its low abundance and lack of information on its metabolism. We developed a DAPI fluorescence-based assay to quantify the low levels of polyP present in mammalian cell lines and tissues, detecting an enrichment of polyP in the mitochondria compared with the nucleus and post-mitochondrial fraction. Mitochondrial polyP synthesis was found to depend on active FoF1 ATP synthase and an intact proton gradient across the inner mitochondrial membrane. Additionally, orthophosphate (Pi) is essential for mitochondrial polyP production, and ATP enhances Pi-driven polyP synthesis in isolated mitochondria. We discovered that the inositol pyrophosphate 5-InsP₇, synthesized by IP6K1, regulates mitochondrial polyP levels. Mice and cells deficient in IP6K1 showed a significant reduction in mitochondrial polyP synthesis compared with wild type controls. Cells lacking IP6K1 also showed impaired mitochondrial respiration. The expression of active IP6K1, but not its catalytically inactive form, restored mitochondrial polyP synthesis in IP6K1 deficient cells, but mitochondrial respiration was rescued by expression of either active or inactive IP6K1. These data show that IP6K1 regulates mitochondrial function and polyP production both through the synthesis of 5-InsP₇ and via a catalytic activity-independent mechanism. Our findings uncover a link between 5-InsP₇, an energy sensor, and polyP, an energy store, in the regulation of mammalian mitochondrial homeostasis.