Abstract
Mitochondrial Ca2+ uptake is crucial for the regulation of the rate of oxidative phosphorylation1, the modulation of spatio-temporal cytosolic Ca2+ signals2,3,4 and apoptosis5. Although the phenomenon of mitochondrial Ca2+ sequestration, its characteristics and physiological consequences have been convincingly reported6,7, the actual protein(s) involved in this process are unknown. Here, we show that the uncoupling proteins 2 and 3 (UCP2 and UCP3) are essential for mitochondrial Ca2+ uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca2+ sequestration in response to cell stimulation under physiological conditions — observations supported by isolated liver mitochondria of Ucp2−/− mice lacking ruthenium red-sensitive Ca2+ uptake. Our results reveal a novel molecular function for UCP2 and UCP3, and may provide the molecular mechanism for their reported effects8,9,10. Moreover, the identification of proteins fundemental for mitochondrial Ca2+ uptake expands our knowledge of the physiological role for mitochondrial Ca2+ sequestration.
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Change history
16 October 2008
In the version of this article initially published, the third and fifth histogram columns in the right panel of Fig. 2a were incorrectly labelled. The third column should have been siUCP3 and the fifth should have been siUPC2. These errors have been corrected in the HTML and PDF versions of the article.
References
Jouaville, L. S., Pinton, P., Bastianutto, C., Rutter, G. A. & Rizzuto, R. Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming. Proc. Natl Acad. Sci. USA 96, 13807–13812 (1999).
Malli, R., Frieden, M., Trenker, M. & Graier, W. F. The role of mitochondria for Ca2+ refilling of the ER. J. Biol. Chem. 280, 12114–12122 (2005).
Hoth, M., Button, D. C. & Lewis, R. S. Mitochondrial control of calcium-channel gating: a mechanism for sustained signaling and transcriptional activation in T lymphocytes. Proc. Natl Acad. Sci. USA 97, 10607–10612 (2000).
Parekh, A. B. Slow feedback inhibition of calcium release-activated calcium current by calcium entry. J. Biol. Chem. 273, 14925–14932 (1998).
Gunter, T. E., Buntinas, L., Sparagna, G., Eliseev, R. & Gunter, K. Mitochondrial calcium transport: mechanisms and functions. Cell Calcium 28, 285–296 (2000).
Duchen, M. R. Mitochondria and Ca2+ in cell physiology and pathophysiology. Cell Calcium 28, 339–348 (2000).
Kirichok, Y., Krapivinsky, G. & Clapham, D. E. The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427, 360–364 (2004).
Brand, M. D. & Esteves, T. C. Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3. Cell Metab. 2, 85–93 (2005).
Jezek, P., Zackova, M., Ruzicka, M., Skobisova, E. & Jaburek, M. Mitochondrial uncoupling proteins--facts and fantasies. Physiol. Res. 53, S199–S211 (2004).
Krauss, S., Zhang, C. Y. & Lowell, B. B. The mitochondrial uncoupling-protein homologues. Nature Rev. Mol. Cell Biol. 6, 248–261 (2005).
Ricquier, D. & Bouillaud, F. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem. J. 345, 161–179 (2000).
Brand, M. D. et al. Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic. Biol. Med. 37, 755–767 (2004).
Dejean, L., Camara, Y., Sibille, B., Solanes, G. & Villarroya, F. Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis. J. Cell Physiol. 201, 294–304 (2004).
Krauss, S. et al. Superoxide-mediated activation of uncoupling protein 2 causes pancreatic β cell dysfunction. J. Clin. Invest. 112, 1831–1842 (2003).
Harper, M. E. et al. Decreased mitochondrial proton leak and reduced expression of uncoupling protein 3 in skeletal muscle of obese diet-resistant women. Diabetes 51, 2459–2466 (2001).
Edgell, C. J., McDonald, C. C. & Graham, J. B. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl Acad. Sci. USA 80, 3734–3737 (1983).
Nagai, T., Sawano, A., Park, E. S. & Miyawaki, A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl Acad. Sci. USA 98, 3197–3202 (2001).
Vidal-Puig, A. J. et al. Energy metabolism in uncoupling protein 3 gene knockout mice. J. Biol. Chem. 275, 16258–16266 (2000).
Yoshitomi, H., Yamazaki, K. & Tanaka, I. Cloning of mouse uncoupling protein 3 cDNA and 5′-flanking region, and its genetic map. Gene 215, 77–84 (1998).
Malli, R. et al. Sustained Ca2+ transfer across mitochondria is essential for mitochondrial Ca2+ buffering, store-operated Ca2+ entry, and Ca2+ store refilling. J. Biol. Chem. 278, 44769–44779 (2003).
Storrie, B. & Madden, E. A. Isolation of subcellular organelles. Methods Enzymol. 182, 203–225 (1990).
Daum, G., Bohni, P. C. & Schatz, G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem. 257, 13028–13033 (1982).
Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).
Zoratti, C., Kipmen-Korgun, D., Osibow, K., Malli, R. & Graier, W. F. Anandamide initiates Ca2+ signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells. Br. J. Pharmacol. 140, 1351–1362 (2003).
Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E. & Fiskum, G. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl Acad. Sci. USA 93, 9893–9898 (1996).
Vergun, O. & Reynolds, I. J. Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+. Biophys. J. 87, 3585–3593 (2004).
Palmer, A. E., Jin, C., Reece, J. C. & Tsien, R. Y. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc. Natl Acad. Sci. USA 101, 17404–17409 (2003).
Osibow, K., Malli, R., Kostner, G. M. & Graier, W. F. A new type of non-Ca2+-buffering apo(a)-based fluorescent indicator for intraluminal Ca2+ in the endoplasmic reticulum. J. Biol. Chem. 281, 5017–5025 (2006).
Schaeffer, G. et al. Intercellular signalling within vascular cells under high D-glucose involves free radical-triggered tyrosine kinase activation. Diabetologia 46, 773–783 (2003).
Malli, R., Frieden, M., Osibow, K. & Graier, W. F. Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation. J. Biol. Chem. 278, 10807–10815 (2003).
Acknowledgements
We thank: B. B. Lowell (Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA) for providing the Ucp2−/− and Ucp3−/− mice; M. D. Brand (MRC Dunn Human Nutrition Unit, Cambridge, UK) for the yeast strains expressing UCP2 and UCP3; A. Miyawaki (Riken,Wako, Saitama, Japan) for mitochondria-targeted ratiometric pericam; C. J. S. Edgell (University of North Carolina, Chapel Hill, NC) for the EA.hy926 cells; T. Pozzan (University Padova, Padova, Italy) for mitochondria-targeted DsRed; R. Tsien (University of California/San Diego, CA) for D1ER and YC4er; and R. Rizzuto (University Ferrara, Ferrara, Italy) for the luciferase construct. The excellent technical assistance of B. Petschar and A. Schreilechner, and the help of K. Osibow in designing cloning strategies are highly appreciated by the authors. We thank N. Demaurex and M. Frieden (University of Geneva, Geneva, Switzerland) for critical review of the manuscript. This work was supported by the Austrian Science Funds (P16860-B9; F3010-B05) and the Franz-Lanyar-Stiftung. The Institute of Molecular Biology and Biochemistry was supported by the infrastructure program of the Austrian ministry of education, science and culture.
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All authors designed this work. M.T. cloned all constructs, designed siRNAs and performed western blots, tissue isolation and Ca2+ measurements. I.F. isolated single mitochondria. S.L.-F. was responsible for breeding the knockout mice. R.M. performed the image analyses and ATP measurements and, together with W.F.G., who wrote the paper, initiated and guided this study. All authors discussed the results and commented on the manuscript.
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Trenker, M., Malli, R., Fertschai, I. et al. Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat Cell Biol 9, 445–452 (2007). https://doi.org/10.1038/ncb1556
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DOI: https://doi.org/10.1038/ncb1556
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