Mitochondrial NAD⁺ gradient sustained by membrane potential and transport

SLC25A51 is required for the replenishment of free nicotinamide adenine dinucleotide (oxidized form) (NAD⁺) into mammalian mitochondria. However, it is not known how SLC25A51 imports this anionic molecule to sustain elevated NAD⁺ concentrations in the matrix. Understanding this would reveal regulatory mechanisms used to maintain critical bioenergetic gradients for cellular respiration, oxidative mitochondrial reactions, and mitochondrial adenosine triphosphate (ATP) production. In this work, mutational analyses and localized NAD⁺ biosensors revealed that the mitochondrial membrane potential (ΔΨm) works in concert with charged residues in the carrier's inner pore to enable sustained import of NAD⁺ against its electrochemical gradient into the matrix. Dissipation of the ΔΨm or mutation of select residues in SLC25A51 led to equilibration of NAD⁺ from the matrix. Corroborating data were obtained with the structurally distinct mitochondrial NAD⁺ carrier from Saccharomyces cerevisiae (ScNdt1p) and mitochondrial ATP transport suggesting a shared mechanism of charge compensation and electrogenic transport in these mitochondrial carrier family members.