Interplay of Mg²⁺, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg²⁺ in cell respiration

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg ²⁺ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using ³¹P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore ( Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg ²⁺ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg ²⁺ in the mitochondrial matrix, where [Mg ²⁺] is tenfold higher. In contrast, owing to a much higher affinity for Mg ²⁺, ATP is mostly complexed by Mg ²⁺ in both compartments. Mg ²⁺ starvation used to alter cytosolic and mitochondrial [Mg ²⁺] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg ²⁺ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. Significance The respiration of heterotrophic cells, where most of the ATP demand is met by mitochondrial oxidative phosphorylation, is generally thought to be regulated either by the ATP/ADP ratio and/or energy charge or by nucleotide concentration. The way in which ADP and ATP may directly mediate respiration remains unclear, however. Furthermore, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP-synthase, Mg ²⁺ compartmentation must be known. For this purpose, we performed simultaneous measurements of free and Mg-complexed nucleotides and Mg ²⁺ in the cytosol and mitochondrial matrix using NMR-based techniques. Physiological alterations induced by Mg starvation helped unravel the key role of cyt