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      Leucine Zipper EF Hand-containing Transmembrane Protein 1 (Letm1) and Uncoupling Proteins 2 and 3 (UCP2/3) Contribute to Two Distinct Mitochondrial Ca 2+ Uptake Pathways*

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          Cytosolic Ca 2+ signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca 2+ domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca 2+/H + antiporter that achieved mitochondrial Ca 2+ sequestration at small Ca 2+ increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca 2+ uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca 2+ but strongly diminished the transfer of entering Ca 2+ into mitochondria, subsequently, resulting in a reduction of store-operated Ca 2+ entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca 2+ signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca 2+ uptake at low Ca 2+ conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca 2+ uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca 2+ uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca 2+ uptake pathways in intact endothelial cells.

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          Most cited references 41

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          MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake

          Mitochondrial calcium uptake plays a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients, and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here, we utilize an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics, and organelle proteomics. RNA interference against 13 top candidates highlighted one gene that we now call mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the organelle’s inner membrane and has two canonical EF hands that are essential for its activity, suggesting a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high capacity mitochondrial calcium entry. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.
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            Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.

            The ER-mitochondrial junction provides a local calcium signaling domain that is critical for both matching energy production with demand and the control of apoptosis. Here, we visualize ER-mitochondrial contact sites and monitor the localized [Ca(2+)] changes ([Ca(2+)](ER-mt)) using drug-inducible fluorescent interorganelle linkers. We show that all mitochondria have contacts with the ER, but plasma membrane (PM)-mitochondrial contacts are less frequent because of interleaving ER stacks in both RBL-2H3 and H9c2 cells. Single mitochondria display discrete patches of ER contacts and show heterogeneity in the ER-mitochondrial Ca(2+) transfer. Pericam-tagged linkers revealed IP(3)-induced [Ca(2+)](ER-mt) signals that exceeded 9 microM and endured buffering bulk cytoplasmic [Ca(2+)] increases. Altering linker length to modify the space available for the Ca(2+) transfer machinery had a biphasic effect on [Ca(2+)](ER-mt) signals. These studies provide direct evidence for the existence of high-Ca(2+) microdomains between the ER and mitochondria and suggest an optimal gap width for efficient Ca(2+) transfer. 2010 Elsevier Inc. All rights reserved.
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              Circularly permuted green fluorescent proteins engineered to sense Ca2+.

               A Sawano,  J. Park,  T Nagai (2001)
              To visualize Ca(2+)-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named "pericam," was fluorescent and its spectral properties changed reversibly with the amount of Ca(2+), probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash-pericam" became brighter with Ca(2+), whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca(2+)-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca(2+) dynamics, such as Ca(2+) oscillations in the cytosol and the nucleus. Ca(2+) imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca(2+) ions across the nuclear envelope. Then, free Ca(2+) concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca(2+) transients caused rapid changes in the concentration of mitochondrial Ca(2+). Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca(2+)-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.

                Author and article information

                J Biol Chem
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                12 August 2011
                25 May 2011
                25 May 2011
                : 286
                : 32
                : 28444-28455
                From the []Institute of Molecular Biology and Biochemistry, Molecular and Cellular Physiology Research Unit, Center of Molecular Medicine, Medical University Graz, Harrachgasse 21/III, 8010 Graz, Austria and
                the [§ ]Precursory Research for Embryonic Science, Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
                Author notes
                [1 ] To whom correspondence should be addressed: Institute of Molecular Biology and Biochemistry, Molecular and Cellular Physiology Research Unit, Center of Molecular Medicine, Medical University Graz, Harrachgasse 21/III, 8010 Graz, Austria. Tel.: 43-316-380-7560; Fax: 43-316-380-9615; E-mail: wolfgang.graier@ .
                © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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