The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux

The NADPH/NADP+ redox couple is central to metabolism and redox signalling. NADP redox state is differentially regulated by distinct enzymatic machineries at the subcellular compartment level. Nonetheless, a detailed understanding of subcellular NADP redox dynamics is limited by the availability of appropriate tools. Here, we introduce NAPstars, a family of genetically encoded, fluorescent protein-based NADP redox state biosensors. NAPstars offer real-time, specific, pH-resistant measurements, across a broad-range of NADP redox states, with subcellular resolution. We establish NAPstar measurements in yeast, plants and mammalian cell models, revealing a conserved robustness of cytosolic NADP redox homeostasis. NAPstars uncovered NADP redox oscillations linked to the cell cycle in yeast and illumination- and hypoxia-dependent NADP redox changes in plant leaves. By selectively impairing the glutathione and thioredoxin anti-oxidative pathways under acute oxidative challenge, NAPstars demonstrated an unexpected role for the glutathione system as the primary mediator of anti-oxidative electron flux that is conserved across eukaryotic kingdoms.