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SLC25A23 augments mitochondrial Ca2+ uptake, interacts with MCU, and induces oxidative stress–mediated cell death

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      Abstract

      Knockdown of SLC25A23 decreases mitochondrial Ca 2+ uptake, and SLC25A23 interacts with MCU and MICU1, components of mitochondrial Ca 2+ uniporter. Expression of SLC25A23 EF-hand-domain mutants has a dominant-negative phenotype of reduced mitochondrial Ca 2+ uptake. It also attenuates basal ROS and oxidant-induced ATP decline and cell death.

      Abstract

      Emerging findings suggest that two lineages of mitochondrial Ca 2+ uptake participate during active and resting states: 1) the major eukaryotic membrane potential–dependent mitochondrial Ca 2+ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca 2+ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca 2+ accumulation are unclear. Solute carriers—solute carrier 25A23 (SLC25A23), SLC25A24, and SLC25A25—represent a family of EF-hand–containing mitochondrial proteins that transport Mg-ATP/Pi across the inner membrane. RNA interference–mediated knockdown of SLC25A23 but not SLC25A24 and SLC25A25 decreases mitochondrial Ca 2+ uptake and reduces cytosolic Ca 2+ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand–domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca 2+ uptake. In addition, SLC25A23 interacts with mitochondrial Ca 2+ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing I MCU. In addition, SLC25A23 knockdown lowers basal mROS accumulation, attenuates oxidant-induced ATP decline, and reduces cell death. Further, reconstitution with short hairpin RNA–insensitive SLC25A23 cDNA restores mitochondrial Ca 2+ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca 2+ influx.

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      Ensembl 2013

      The Ensembl project (http://www.ensembl.org) provides genome information for sequenced chordate genomes with a particular focus on human, mouse, zebrafish and rat. Our resources include evidenced-based gene sets for all supported species; large-scale whole genome multiple species alignments across vertebrates and clade-specific alignments for eutherian mammals, primates, birds and fish; variation data resources for 17 species and regulation annotations based on ENCODE and other data sets. Ensembl data are accessible through the genome browser at http://www.ensembl.org and through other tools and programmatic interfaces.
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        A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter

        Mitochondrial Ca2+ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival 1 , but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter 2 , was still unknown. We have identified in silico a protein (denominated MCU) that shares tissue distribution with MICU1, a recently characterized uniporter regulator 3 , coexists with uniporter activity in phylogeny and includes two trasmembrane domains in the sequence. siRNA silencing of MCU in HeLa cells drastically reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the [Ca2+]mt rise evoked by IP3-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein exhibited channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively-charged residues of the putative pore forming region were replaced, had no channel activity and reduced agonist-dependent [Ca2+]mt transients when overexpressed in HeLa cells. Overall, these data demonstrate that the identified 40 kDa protein is the channel responsible for Ruthenium Red-sensitive mitochondrial Ca2+ uptake, thus providing molecular basis for this process of utmost physiological and pathological relevance.
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          Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.

          Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter. Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1. All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call 'mitochondrial calcium uniporter' (MCU). MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca(2+) uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter. Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca(2+) uniporter.
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            Author and article information

            Affiliations
            aDepartment of Biochemistry, Temple University, Philadelphia, PA 19140
            bCenter for Translational Medicine, Temple University, Philadelphia, PA 19140
            Cornell University
            Author notes
            1Address correspondence to: Muniswamy Madesh ( madeshm@ 123456temple.edu ).
            Contributors
            Role: Monitoring Editor
            Journal
            Mol Biol Cell
            Mol. Biol. Cell
            molbiolcell
            mbc
            Mol. Bio. Cell
            Molecular Biology of the Cell
            The American Society for Cell Biology
            1059-1524
            1939-4586
            15 March 2014
            : 25
            : 6
            : 936-947
            24430870 3952861 E13-08-0502 10.1091/mbc.E13-08-0502
            © 2014 Hoffman et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License ( http://creativecommons.org/licenses/by-nc-sa/3.0).

            “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

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