Release of Ca2+ and Mg2+ from yeast mitochondria is stimulated by increased ionic strength.

Divalent cations are required for many essential functions of mitochondrial metabolism. Yet the transporters that mediate the flux of these molecules into and out of the mitochondrion remain largely unknown. Previous studies in yeast have led to the molecular identification of a component of the major mitochondrial electrophoretic Mg2+ uptake system in this organism as well as a functional mammalian homolog. Other yeast mitochondrial studies have led to the characterization of an equilibrative fatty acid-stimulated Ca2+ transport activity. To gain a deeper understanding of the regulation of mitochondrial divalent cation levels we further characterized the efflux of Ca2+ and Mg2+ from yeast mitochondria. When isolated mitochondria from the yeast Saccharomyces cerevisiae were suspended in a salt-based suspension medium, Ca2+ and Mg2+ were released from the matrix space. Release did not spontaneously occur in a non-ionic mannitol media. When energized mitochondria were suspended in a mannitol medium in the presence of Ca2+ they were able to accumulate Ca2+ by the addition of the electrogenic Ca2+ ionophore ETH-129. However, in a KCl or choline Cl medium under the same conditions, they were unable to retain the Ca2+ that was taken up due to the activation of the Ca2+ efflux pathway, although a substantial membrane potential driving Ca2+ uptake was maintained. This Ca2+ efflux was independent of fatty acids, which have previously been shown to activate Ca2+ transport. Endogenous mitochondrial Mg2+ was also released when mitochondria were suspended in an ionic medium, but was retained in mitochondria upon fatty acid addition. When suspended in a mannitol medium, metal chelators released mitochondrial Mg2+, supporting the existence of an external divalent cation-binding site regulating release. Matrix space Mg2+ was also slowly released from mitochondria by the addition of Ca2+, respiratory substrates, increasing pH, or the nucleotides ATP, ADP, GTP, and ATP-gamma-S. In isolated yeast mitochondria Ca2+ and Mg2+ release was activated by increased ionic strength. Free nucleotides, metal ion chelators, and increased pH also stimulated release. In yeast cells this release is likely an important mechanism in the regulation of mitochondrial matrix space divalent cation concentrations.