MiP2005: Session 3 - Young Investigator Presentation

Mitochondrial Physiology Network 10.9: 37 (2005) - download pdf


Calcium activation of the malate-aspartate shuttle in tissues expressing distinct isoforms of the mitochondrial aspartate-glutamate carrier.

Laura Contreras1, B Pardo1, T Saheki2, J Satrústegui1

1Departamento de Biología Molecular, CBMSO, Universidad Autónoma de Madrid-C.S.I.C. 28049 Madrid, Spain; 2Dept. Molecular Metabolism Biochemical Genetics, Kagoshima University, Graduate School of Medical and Dental Sciences 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. - lcontreras@cbm.uam.es

    Aralar and citrin are the human isoforms of the aspartate-glutamate carrier (AGC) involved in the NADH Malate-Aspartate shuttle (MAS) for the transfer of reducing equivalents from cytosol to mitochondria [1-4]. Aralar and citrin have several EF-hands motifs in a long amino terminal extension that faces the intermembrane space. We have tested a possible role for extra mitochondrial calcium in activation of MAS, as the step catalyzed by the AGC is the only one irreversible of that pathway. To this end, we have measured the calcium sensitivity of the shuttle reconstituted in isolated mitochondria, in the presence of ruthenium red to inhibit the calcium uniporter. Aralar and citrin have different expression patterns [5], and we have studied the kinetics of calcium activation in tissues with only one isoform. Mitochondria of tissues which express only aralar (namely brain and muscle) exhibit a calcium activation of MAS of 3 fold, with an S0.5 of about 340-230 nM, whereas in liver, where only citrin is present, the increase of activity due to calcium was lower (1.5-fold) with a S0.5 of 100-140 nM. In the heart, where both isoforms are coexpressed, calcium activation is also observed (S0.5 230 nM, 3.5-fold activation), and appears to be due to aralar since it disappears completely in Aralar-deficient mice.

    So far, calcium signalling in mitochondria is believed to take place mainly through calcium uptake in mitochondria across the calcium uniporter (CU), followed by activation of three dehydrogenases (pyruvate, isocitrate and a-ketoglutarate) of the mitochondrial matrix that results in an increased production of mitochondrial NADH. Calcium uptake through CU requires relatively high calcium concentrations [6], higher than those activating MAS. In conclusion, MAS is activated by calcium concentrations smaller than those activating the CU-mitochondrial dehydrogenase pathway, specially through aralar as AGC. Therefore, it is an adequate mechanism to transduce small cytosolic calcium signals to the mitochondria. On the other hand, the very low S0.5 for calcium in liver mitochondria in which citrin is the only AGC suggests that  MAS would be constitutively activated at the normal resting [Ca2+]i.

1.    del Arco A, Satrústegui J (1998) Molecular cloning of Aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain.  J. Biol. Chem. 273:23327-23334.

2.    Kobayashi K, Sinasa DS, Iijima M, Boright AP, Begum L, Lee JR, Yasuda T, Ikeda S, Hirano R, Terazono H, Crackower MA, Kondo I, Tsui LC, Scherer SW, Saheki T (1999) The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein. Nat. Genet. 22: 159-163.

3.    del Arco A, Agudo M., Satrústegui J (2000) Characterization of a second member of the subfamily of calcium-binding mitochondrial carriers expressed in human non-excitable tissues. Biochem. J. 345: 725-732.

4.    Palmieri L, Pardo B, Lasorsa FM, del Arco A, Kobayashi K, Iijima M, Runswick MJ, Walker JE, Saheki T, Satrústegui J (2001) Citrin and aralar1 are Ca2+-stimulated aspartate/glutamate transporters in mitochondria. EMBO J. 20: 5060-5069.

5.    del Arco A, Morcillo J., Martínez-Morales JR, Galián C, Martos V, Bovolenta P, Satrústegui J (2002) Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues. Eur. J. Biochem. 269: 3313-3320.

6.    Gunter TE, Yule DI, Gunter KK, Eliseev RA, Salter JD (2004) Calcium and mitochondria FEBS Lett. 567: 96-102.

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Mitochondrial Physiology