Spatiotemporal biosensor profiling reveals an autonomous mitochondrial NAD+/NMN regulatory network centered on NMNAT3

Nicotinamide adenine dinucleotide (NAD+) and its precursor nicotinamide mononucleotide (NMN) are strictly compartmentalized, yet how individual organelles maintain local metabolic homeostasis remains unresolved. Here, we report FrNADS and FrNMNS1.0, a FRET-based biosensor toolkit that maps NAD+ and NMN dynamics in living cells with subcellular resolution, including the oxidizing lumen of the endoplasmic reticulum. We find that NAD+ recovery in the nucleus following PARP1 activation depends on NAMPT mediated salvage synthesis, while peroxisomes buffer NAD+ via NUDT12 and SLC25A17. In mitochondria, NMNAT3 acts as a NAD+ hydrolase that counterbalances import through SLC25A51; HINT2 functionally enhances this activity. Furthermore, SLC25A48 functions as a critical regulatory node that modulates the compartmental redistribution of the generated NMN. These findings establish a mitochondrial NAD+/NMN regulatory circuit and reveal how organelles independently resolve metabolic stress.