Nat. Cell Biol. 14, 1336–1343 (2012); published online 25 November 2012; corrected after print 10 June 2015 In the version of this Letter originally published, a key funding source was omitted from the Acknowledgements. César Cárdenas's credit should have read 'C.C. was supported by the Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT) grant #1120443 and an award from the American Heart Association'.

Emerging findings suggest that two lineages of mitochondrial Ca2+ uptake participate during active and resting states: 1) the major eukaryotic membrane potential-dependent mitochondrial Ca2+ uniporter and 2) the evolutionarily conserved exchangers and solute carriers, which are also involved in ion transport. Although the influx of Ca2+ across the inner mitochondrial membrane maintains metabolic functions and cell death signal transduction, the mechanisms that regulate mitochondrial Ca2+ 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 Ca2+ uptake and reduces cytosolic Ca2+ clearance after histamine stimulation. Ectopic expression of SLC25A23 EF-hand-domain mutants exhibits a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. In addition, SLC25A23 interacts with mitochondrial Ca2+ uniporter (MCU; CCDC109A) and MICU1 (CBARA1) while also increasing IMCU. 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 Ca2+ uptake and superoxide production. These findings indicate that SLC25A23 plays an important role in mitochondrial matrix Ca2+ influx.

Dysregulation of mitochondrial Ca2+-dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca2+ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca2+ transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca2+ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca2+ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in EF hand, (D676A D688K)LETM1 mutant-overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS-derived fibroblasts, the mitochondrial Ca2+ uniporter components MCU, MCUR1, and MICU1 remain unaltered. In addition, the MCU mitoplast patch-clamp current (I-MCU) was largely unaffected in LETM1-knockdown cells. Silencing of LETM1 also impaired basal mitochondrial oxygen consumption, possibly via complex IV inactivation and ATP production. Remarkably, LETM1 knockdown also resulted in increased reactive oxygen species production. Further, LETM1 silencing promoted AMPK activation, autophagy, and cell cycle arrest. Reconstitution of LETM1 or antioxidant overexpression rescued mitochondrial Ca2+ transport and bioenergetics. These findings reveal the role of LETM1-dependent mitochondrial Ca2+ flux in shaping cellular bioenergetics.Doonan, P J., Chandramoorthy, H. C., Hoffman, N. E., Zhang, X., Cardenas, C., Shanmughapriya, S., Rajan, S., Vallem, S., Chen, X., Foskett, J. K., Cheung, J. Y., Houser, S. R., Madesh, M. LETM1-dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation.

Microbial infection triggers assembly of inflammasome complexes that promote caspase-1-dependent antimicrobial responses. Inflammasome assembly is mediated by members of the nucleotide binding domain leucine-rich repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through NLRC4, whereas NLRP3 is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by down-regulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation after Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome. We therefore screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial reactive oxygen species and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11-dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, our data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence.

Mitochondrial Ca(2+) (Ca(2+)(m)) uptake is mediated by an inner membrane Ca(2+) channel called the uniporter. Ca(2+) uptake is driven by the considerable voltage present across the inner membrane (ΔΨ(m)) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca(2+) concentration is maintained five to six orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here, we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca(2+)](m) under basal conditions. In its absence, mitochondria become constitutively loaded with Ca(2+), triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca(2+) threshold for Ca(2+)(m) uptake without affecting the kinetic properties of MCU-mediated Ca(2+) uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca(2+)(m) uptake that is essential to prevent [Ca(2+)](m) overload and associated stress.

The c-Myc oncoprotein regulates greater than 15% of the human transcriptome and a limited number of microRNAs. Here we establish that in a human B-lymphoid cell line Myc-repressed, but not Mycstimulated, genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17~92 cluster. Notably, gene set enrichment analysis (GSEA) demonstrates that miR-17~92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17~92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17~92 expression was necessary to sustain phosphorylation of SYK and BLNK upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux and elevated levels of Myc itself. Notably, inhibition of the miR-17~92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop.

evidently increased, suggesting the acidification between the membranesurface space. Additionally, the degree of the acidification was proportional to the concentration of uncaged ATP ranging from 0.2 mM to 0.7 mM. These results suggest that we successfully detected the proton pumping activity sensitively and quantitatively. As many kinds of Caged compounds and ion sensitive fluorescent dyes are commercially available, this technique can be applied to other ion pumps and transporters.

The c-Myc oncoprotein regulates >15% of the human transcriptome and a limited number of microRNAs (miRNAs). Here, we establish that in a human B-lymphoid cell line, Myc-repressed, but not Myc-stimulated, genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17~92 cluster. Notably, gene set enrichment analysis demonstrates that miR-17∼92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17∼92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17~92 expression was necessary to sustain phosphorylation of spleen tyrosine kinase (SYK) and the B-cell linker protein (BLNK) upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux and elevated levels of Myc itself. Notably, inhibition of the miR-17~92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop.

Ca2+ flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca2+ signals and cell death pathways. Mitochondrial Ca2+ uptake occurs at regions of close apposition with intracellular Ca2+ release sites, driven by the inner membrane voltage generated by oxidative phosphorylation (OXPHOS) and mediated by a Ca2+ selective ion channel (MiCa) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was identified as the likely ion-conducting pore. Mitochondrial matrix Ca2+ concentration is maintained 5-6 orders of magnitude lower than its thermodynamic equilibrium level, but the molecular mechanisms for how this is achieved are not clear. We found that MICU1, a mitochondrial protein previously suggested to be required for uniporter-mediated Ca2+ uptake, is instead required to preserve normal [Ca2+]m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca2+, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with MCU and sets a Ca2+ threshold for Ca2+m uptake without affecting kinetic properties of MCU-mediated Ca2+ uptake, a regulation that requires both functional Ca2+ binding EF hands in MICU1. Thus, MICU1 is a gatekeeper of MCU-mediated Ca2+m uptake that is essential to prevent [Ca2+]m overload and associated stress. Additionally, we have also identified CCDC90A, hereafter referred to as MCUR1 (Mitochondrial Calcium Uniporter Regulator 1), as an inner mitochondrial membrane protein required for MCU-dependent mitochondrial Ca2+ uptake. MCUR1 binds to MCU and regulates ruthenium red-sensitive MCU-dependent Ca2+ uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca2+ uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts OXPHOS, lowers cellular ATP, and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 and MICU1 are critical components of a mitochondrial uniporter channel complex required for mitochondrial Ca2+ uptake and maintenance of normal cellular bioenergetics.

Regulation of mitochondrial Ca2+‐dependent cellular bioenergetics has been implicated in a variety of pathophysiological settings including neurodegeneration, myocardial infarction and immunity. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter; however characterization of the functional role of LETM1‐mediated Ca2+ transfer has yet to be studied. Here we demonstrate that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake and results in bioenergetic collapse. We observed that mitochondrial Ca2+ uptake is impaired in shRNA‐mediated LETM1 knockdown and ΔEF hand LETM1 and D676AD688KLETM1 overexpression cells. Knockdown of LETM1 promotes LC3 positive multilamellar vesicle formation, indicative of autophagy induction. Furthermore, cellular bioenergetic parameters including ATP and oxygen consumption were reduced. In contrast, cellular NADH and mitochondrial membrane potential were unaltered in both control and shRNA LETM1 cells. Interestingly, knockdown of LETM1 by siRNA in primary neonatal cardiomyocytes resulted in a remarkable change in mitochondrial morphology and reduced Ca2+ uptake. We also observed that LETM1 knockdown results in increased ROS production. Our results represent the first time that LETM1 has been linked to cellular bioenergetics through regulation of mitochondrial Ca2+.Supported by NIH R01 HL086699, HL086699‐01A2S1, 1S10RR027327‐01

Nat. Cell Biol. 14, 1336–1343 (2012); published online 25th November 2012; corrected online 30th November 2012 In the version of this Letter that was originally published, the affiliation of Jordi Molgó was erroneous. The correct affiliation is: CNRS, Institut de Neurobiologie Alfred Fessard FRC2118, Laboratoire de Neurobiologie et Développement, UPR 3294, 91198 Gif-sur-Yvette cedex, France.

Mitochondrial membrane potential loss has severe bioenergetic consequences and contributes to many human diseases including myocardial infarction, stroke, cancer, and neurodegeneration. However, despite its prominence and importance in cellular energy production, the basic mechanism whereby the mitochondrial membrane potential is established remains unclear. Our studies elucidate that complex II-driven electron flow is the primary means by which the mitochondrial membrane is polarized under hypoxic conditions and that lack of the complex II substrate succinate resulted in reversible membrane potential loss that could be restored rapidly by succinate supplementation. Inhibition of mitochondrial complex I and F(0)F(1)-ATP synthase induced mitochondrial depolarization that was independent of the mitochondrial permeability transition pore, Bcl-2 (B-cell lymphoma 2) family proteins, or high amplitude swelling and could not be reversed by succinate. Importantly, succinate metabolism under hypoxic conditions restores membrane potential and ATP levels. Furthermore, a reliance on complex II-mediated electron flow allows cells from mitochondrial disease patients devoid of a functional complex I to maintain a mitochondrial membrane potential that conveys both a mitochondrial structure and the ability to sequester agonist-induced calcium similar to that of normal cells. This finding is important as it sets the stage for complex II functional preservation as an attractive therapy to maintain mitochondrial function during hypoxia.

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.

Ethanol intoxication stimulates the production of proinflammatory cytokines, increases the formation of reactive oxygen species, and induces mitochondrial impairment. However, information is limited as to the exact sequence and components involved in ethanol-induced hepatotoxicity. Acute ethanol exposure enhances mitochondrial superoxide (O(2)(*-)) production and impairs mitochondrial Ca(2+) handling. In turn, O(2)(*-) facilitates cytochrome c release and mitochondrial membrane potential loss that is not dependent upon H(2)O(2) and divalent cations and requires Bak in a Bax-independent fashion. Furthermore, triggering of Bak's proapoptotic activity requires the cytosolic presence of Bid, a BH3-only protein that is processed by the initiator caspase-2. Together, these studies identify an O(2)(*-)-driven, caspase-initiated apoptotic pathway that selectively involves the Bcl-2 family proteins Bid and Bak. This pathway manifests itself during chronic ethanol consumption in aged animals and identifies caspase-2, Bid, and Bak as essential mediators of O(2)(*-)-induced apoptosis that may prove effective targets for the development of therapeutics to treat alcoholic liver disease.