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. 2011 Jun 19;476(7360):341-5.
doi: 10.1038/nature10234.

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter

Joshua M Baughman  1 , Fabiana Perocchi, Hany S Girgis, Molly Plovanich, Casey A Belcher-Timme, Yasemin Sancak, X Robert Bao, Laura Strittmatter, Olga Goldberger, Roman L Bogorad, Victor Koteliansky, Vamsi K Mootha
Affiliations

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter

Joshua M Baughman et al. Nature. .

Abstract

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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Figures

Figure 1
Figure 1. Integrative genomics predicts MCU to be functionally related to MICU1
a–c, Phylogenetic profile neighbours (a), RNA co-expression neighbours (b) and protein co-expression neighbours (c) of MICU1. Hamming distances between phylogenetic profiles were computed genome-wide for all 20,000 mammalian genes across 500 fully sequenced organisms. Genes co-expressed with MICU1 were computed genome-wide by Pearson correlation using a mouse atlas of 81 tissues. Protein expression correlation with MICU1 was analysed for all mitochondrial proteins across 14 mouse tissues. d, Coimmunoprecipitation of MICU1 and MCU. HEK-293 cells stably expressing MICU1–V5 or MCU–V5 were transfected with MICU1–GFP, MCU–GFP, MFRN2–GFP or UCP2–GFP. Cell lysates were incubated with anti-V5 antibody, immunoprecipitates were resolved on SDS–PAGE, and input lysates and immunoprecipitates were blotted with anti-V5 (top) or anti-GFP (bottom) antibodies. Data are representative of three independent experiments. IP, immunoprecipitation; GF, green fluorescent protein; IB, immunoblot.
Figure 2
Figure 2. MCU is required for mitochondrial Ca2+ uptake in cultured cells and in purified mouse liver mitochondria
a, Representative luminescence measurements of a mitochondrial aequorin Ca2+ reporter after histamine stimulation in HeLa cells expressing sh-LACZ, sh-MCU, or a combination of sh-MCU and an RNAi-resistant cDNA for MCU (mean ± s.e.m., n = 10 traces). Inset shows statistical analysis of the maximal luminescence (mean ± s.d., n= 10 traces, *P<0.001). a.u., arbitrary units. b, Relationship between MCU mRNA expression and histamine-induced mitochondrial Ca2+ uptake (maximal aequorin luminescence) recorded from five independent shRNAs targeting MCU and normalized to sh-LACZ (mean ± s.d., n= 3). c, Representative traces of Ca2+ uptake in digitonin-permeabilized sh-MCU HEK-293 cells or sh-LACZ control cells after addition of 50 μM final concentration of CaCl2. Inset reports linear fits of uptake kinetics between 15 and 20 s, normalized to sh-LACZ (mean ± s.d., n = 3, *P <0.001). Ca2+ was measured with Calcium Green-5N. d, In vitro dose–response of a selected siRNA duplex targeting mouse MCU. Relative expression of MCU mRNA in livers following weekly injections of si-MCU or si-LUC for 3 weeks, normalized to expression in PBS-treated mice. Immunoblot analysis of liver mitochondria isolated from mice treated with si-MCU or control si-LUC using antibodies against MCU and ATP5A1 as a loading control. e, Oxygen consumption measurements of isolated mitochondria in a well-stirred cuvette. Glutamate and malate (G/M), ADP, uncoupler (carbonyl cyanide m-chlorophenyl hydrazone, CCCP), antimycin (AM) were added at indicated time points. Respiratory control ratio (RCR) and ADP:O ratio (P:O) were computed from three separate mice for each group. f, Mitochondrial membrane potential (ψm) measured by tetramethyl rhodamine methyl ester (TMRM) in isolated liver mitochondria. g, Ca2+ uptake kinetics in energized liver mitochondria following the addition of 50 μM final CaCl2. Extra-mitochondrial Ca2+ was measured with Calcium Green-5N (mean ± s.e.m., n = 3 mice). Traces depicted in e and f are representative of measurements made from three independent mouse experiments performed on separate days.
Figure 3
Figure 3. MCU is oligomeric and resides in the mitochondrial inner membrane as a larger complex
a, Confocal imaging of MCU–GFP co-expressed with mitochondria-targeted HcRed (Mito-HcRed) in HeLa cells. b, Immunoblot analysis of HeLa whole-cell lysate (WCL), cytosol (Cyto) or crude mitochondrial fractions (Mito), using antibodies against MCU, HSP60 (matrix protein, also known as HSPD1), or ACTB (cytosol). c, Immunoblot analysis of soluble (supernatant) and insoluble (pellet) fractions following alkaline carbonate extraction of mitochondrial fractions from HEK-293 cells expressing MCU–V5. Immunoblot analysis was performed using antibodies against V5, COII (integral inner membrane protein) and CYCS (soluble intermembrane space protein). d, Immunoblot analysis after proteinase K (PK) treatment of MCU–V5-expressing HEK-293 mitoplasts for indicated times. e, Anti-V5 immunoprecipitations performed as in Fig. 1d using lysates from HEK-293 cells stably expressing MCU–V5 and transiently transfected with MCU–GFP, UCP2–GFP, or MFRN2–GFP. f, Blue native PAGE analysis of mitochondrial fractions from HeLa cells (stably expressing sh-LACZ or sh-MCU, left panel) or from livers of mice (si-LUC or si-MCU, right panel) and immunoblotted for MCU. ATP5A1 is used as a loading control.
Figure 4
Figure 4. Impact of point mutations on MCU activity and its sensitivity to Ru360
a, Schematic of MCU topology across the mitochondrial inner membrane and a multiple sequence alignment of the linker sequence containing a DIME motif. TM1 and TM2 are two transmembrane domains. b, Ca2+ uptake in permeabilized sh-MCU HEK-293 cells transiently expressing MCU mutants. Inset reports linear fits of uptake kinetics between 15 and 25 s, normalized to sh-LACZ (mean ± s.d., n = 3, *P <0.01) c, Ca2+ uptake in HEK-293 cells transiently expressing wild-type MCU or the S259A mutant, in the presence or absence of 0.5 μM Ru360. Inset reports linear fits of uptake kinetics between 30 and 60 s for Ru360-treated cells, and between 15 and 25 s for untreated cells. Uptake rates are normalized to untreated HEK-293 cells (mean ± s.d., n = 3, *P <0.01).
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