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1.  Release of Ca2+ and Mg2+ from yeast mitochondria is stimulated by increased ionic strength 
BMC Biochemistry  2006;7:4.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2091-7-4
PMCID: PMC1386685  PMID: 16460565
2.  Loss of NAD(H) from swollen yeast mitochondria 
BMC Biochemistry  2006;7:3.
Background
The mitochondrial electron transport chain oxidizes matrix space NADH as part of the process of oxidative phosphorylation. Mitochondria contain shuttles for the transport of cytoplasmic NADH reducing equivalents into the mitochondrial matrix. Therefore for a long time it was believed that NAD(H) itself was not transported into mitochondria. However evidence has been obtained for the transport of NAD(H) into and out of plant and mammalian mitochondria. Since Saccharomyces cerevisiae mitochondria can directly oxidize cytoplasmic NADH, it remained questionable if mitochondrial NAD(H) transport occurs in this organism.
Results
NAD(H) was lost more extensively from the matrix space of swollen than normal, condensed isolated yeast mitochondria from Saccharomyces cerevisiae. The loss of NAD(H) in swollen organelles caused a greatly decreased respiratory rate when ethanol or other matrix space NAD-linked substrates were oxidized. Adding NAD back to the medium, even in the presence of a membrane-impermeant NADH dehydrogenase inhibitor, restored the respiratory rate of swollen mitochondria oxidizing ethanol, suggesting that NAD is transported into the matrix space. NAD addition did not restore the decreased respiratory rate of swollen mitochondria oxidizing the combination of malate, glutamate, and pyruvate. Therefore the loss of matrix space metabolites is not entirely specific for NAD(H). However, during NAD(H) loss the mitochondrial levels of most other nucleotides were maintained. Either hypotonic swelling or colloid-osmotic swelling due to opening of the yeast mitochondrial unspecific channel (YMUC) in a mannitol medium resulted in decreased NAD-linked respiration. However, the loss of NAD(H) from the matrix space was not mediated by the YMUC, because YMUC inhibitors did not prevent decreased NAD-linked respiration during swelling and YMUC opening without swelling did not cause decreased NAD-linked respiration.
Conclusion
Loss of endogenous NAD(H) from isolated yeast mitochondria is greatly stimulated by matrix space expansion. NAD(H) loss greatly limits NAD-linked respiration in swollen mitochondria without decreasing the NAD-linked respiratory rate in normal, condensed organelles. NAD addition can totally restore the decreased respiration in swollen mitochondria. In live yeast cells mitochondrial swelling has been observed prior to mitochondrial degradation and cell death. Therefore mitochondrial swelling may stimulate NAD(H) transport to regulate metabolism during these conditions.
doi:10.1186/1471-2091-7-3
PMCID: PMC1395316  PMID: 16433924

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