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Mitochondria require nicotinamide adenine dinucleotide (NAD + ) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD + transporters have been identified in yeast and plants 1,2 , but their existence in mammals remains controversial 3-5 . Here we demonstrate that mammalian mitochondria can take up intact NAD + , and identify SLC25A51 (also known as MCART1)-an essential 6,7 mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD + transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD + content, impairs mitochondrial respiration, and blocks the uptake of NAD + into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD + levels and restores NAD + uptake into yeast mitochondria lacking endogenous NAD + transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD + into mitochondria.

Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

Mitochondrial dysfunction is a critical step in the pathogenesis of many neurodegenerative diseases. The p32/ C1qbp gene functions as an essential RNA and protein chaperone in mitochondrial translation, and is indispensable for embryonic development. However, little is known about the consequences of mitochondrial dysfunction of p32 deletion in the brain development. Here, we found that mice lacking p32 in the central nervous system (p32cKO mice) showed white matter degeneration accompanied by progressive oligodendrocyte loss, axon degeneration and vacuolation in the mid brain and brain stem regions. Furthermore, p32cKO mice died within 8 weeks of birth. We also found that p32-deficient oligodendrocytes and neurons showed reduced oligodendrocyte differentiation and axon degeneration in primary culture. We show that mitochondrial disruption activates an adaptive program known as the integrated stress response (ISR). Mitochondrial respiratory chain function in oligodendrocytes and neurons is, therefore, essential for myelination and axon maintenance, respectively, suggesting that mitochondrial respiratory chain dysfunction in the central nervous system contributes to leukoencephalopathy.

D -Amino acids are enantiomers of L-amino acids and have recently been recognized as biomarkers and bioactive substances in mammals, including humans. In the present study, we investigated functions of the novel mammalian mitochondrial protein 9030617O03Rik and showed decreased expression under conditions of heart failure. Genomic sequence analyses showed partial homology with a bacterial aspartate/glutamate/hydantoin racemase. Subsequent determinations of all free amino acid concentrations in 9030617O03Rik-deficient mice showed high accumulations of D-glutamate in heart tissues. This is the first time that a significant amount of D-glutamate was detected in mammalian tissue. Further analysis of D-glutamate metabolism indicated that 9030617O03Rik is a D-glutamate cyclase that converts D-glutamate to 5-oxo-D-proline. Hence, this protein is the first identified enzyme responsible for mammalian D-glutamate metabolism, as confirmed in cloning analyses. These findings suggest that D-glutamate and 5-oxo-D-proline have bioactivities in mammals through the metabolism by D-glutamate cyclase.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

Mitochondria are highly dynamic organelles undergoing constant network reorganization and exhibiting stochastic signaling events in the form of mitochondrial flashes (mitoflashes). Here we investigate whether and how mitochondrial network dynamics regulate mitoflash biogenesis and signaling. We found that mitoflash frequency was largely invariant when network fragmentized or redistributed in the absence of mitofusin (Mfn) 1, Mfn2, or Kif5b. However, Opa1 deficiency decreased spontaneous mitoflash frequency due to superimposing changes in respiratory function, whereas mitoflash response to non-metabolic stimulation was unchanged despite network fragmentation. In Drp1- or Mff-deficient cells whose mitochondria hyperfused into a single whole-cell reticulum, the frequency of mitoflashes of regular amplitude and duration was again unaltered, although brief and low-amplitude "miniflashes" emerged because of improved detection ability. As the network reorganized, however, the signal mass of mitoflash signaling was dynamically regulated in accordance with the degree of network connectivity. These findings demonstrate a novel functional role of mitochondrial network dynamics and uncover a magnitude- rather than frequency-modulatory mechanism in the regulation of mitoflash signaling. In addition, our data support a stochastic trigger model for the ignition of mitoflashes.

Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1). Thermogenesis from these adipocytes can combat obesity and diabetes, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders.

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

Reduced expression of the pro-apoptotic protein SMAC (second mitochondria-derived activator of caspase) has been reported to correlate with cancer progression, while its significance and underlying mechanisms are poorly understood. In this study, we investigated the role of SMAC in intestinal tumorigenesis using both human samples and animal models. Decreased SMAC expression was found to correlate with increased cIAP2 expression and higher grades of human colon cancer. In mice, SMAC deficiency significantly increased the incidence and size of colon tumors induced by azoxymethane (AOM)/dextran sulfate sodium salt (DSS), and highly enriched β-catenin hot spot mutations. SMAC deficiency also significantly increased the incidence of spontaneous intestinal polyps in APC(Min/+) mice. Loss of SMAC in mice led to elevated levels of cIAP1 and cIAP2, increased proliferation and activation of the NF-κB p65 subunit in normal and tumor tissues. Unexpectedly, SMAC deficiency had little effect on the incidence of precursor lesions, or apoptosis induced by AOM or DSS, or in established tumors in mice. Furthermore, SMAC knockout enhanced TNFα-mediated NF-κB activation via cIAP2 in HCT 116 colon cancer cells. These results demonstrate an essential and apoptosis-independent function of SMAC in tumor suppression and provide new insights into the biology and targeting of colon cancer.

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt (tq209)). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe-2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.

Mitochondrial calcium uptake has 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 use 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, CBARA1, that we call hereafter 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 mitochondrial inner membrane and has two canonical EF hands that are essential for its activity, indicating a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. 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.

The insulin-like growth factor (IGF-I) signalling pathway is essential for metabolism, cell growth and survival. It induces expression of the mitochondrial pyrimidine nucleotide carrier 1 (PNC1) in transformed cells, but the consequences of this for cell phenotype are unknown. Here we show that PNC1 is necessary to maintain mitochondrial function by controlling mitochondrial DNA replication and the ratio of transcription of mitochondrial genes relative to nuclear genes. PNC1 suppression causes reduced oxidative phosphorylation and leakage of reactive oxygen species (ROS), which activates the AMPK-PGC1alpha signalling pathway and promotes mitochondrial biogenesis. Overexpression of PNC1 suppresses mitochondrial biogenesis. Suppression of PNC1 causes a profound ROS-dependent epithelial-mesenchymal transition (EMT), whereas overexpression of PNC1 suppresses both basal EMT and induction of EMT by TGF-beta. Overall, our findings indicate that PNC1 is essential for mitochondria maintenance and suggest that its induction by IGF-I facilitates cell growth whereas protecting cells from an ROS-promoted differentiation programme that arises from mitochondrial dysfunction.

Juxtaposition between endoplasmic reticulum (ER) and mitochondria is a common structural feature, providing the physical basis for intercommunication during Ca(2+) signalling; yet, the molecular mechanisms controlling this interaction are unknown. Here we show that mitofusin 2, a mitochondrial dynamin-related protein mutated in the inherited motor neuropathy Charcot-Marie-Tooth type IIa, is enriched at the ER-mitochondria interface. Ablation or silencing of mitofusin 2 in mouse embryonic fibroblasts and HeLa cells disrupts ER morphology and loosens ER-mitochondria interactions, thereby reducing the efficiency of mitochondrial Ca(2+) uptake in response to stimuli that generate inositol-1,4,5-trisphosphate. An in vitro assay as well as genetic and biochemical evidences support a model in which mitofusin 2 on the ER bridges the two organelles by engaging in homotypic and heterotypic complexes with mitofusin 1 or 2 on the surface of mitochondria. Thus, mitofusin 2 tethers ER to mitochondria, a juxtaposition required for efficient mitochondrial Ca(2+) uptake.

BIK is the founding member of the BH3-only family pro-apoptotic proteins. BIK is predominantly localized in the ER and induces apoptosis through the mitochondrial pathway by mobilizing calcium from the ER to the mitochondria and remodeling the mitochondrial cristae. BIK-mediated apoptosis is mediated by selective activation of BAX. BIK also induces non-apoptotic cell death in certain cell types by unknown mechanisms. BIK is non-essential for animal development, but appears to be functionally redundant for certain developmental functions with BIM. BIK is implicated in the selection of mature B cells in humans. BIK is a pro-apoptotic tumor suppressor in several human tissues and its expression in cancers is prevented by chromosomal deletions encompassing the Bik locus or by epigenetic silencing. BIK appears to be a critical effector in apoptosis induced by toxins, cytokines and virus infection. Several anti-cancer drugs transcriptionally activate Bik gene expression through transcriptional pathways dependent on factors such as E2F and p53 or by removal of epigenetic marks on the chromatin. BIK appears to be a prominent target for anti-cancer drugs that inhibit proteasomal functions. BIK has also been used as a therapeutic molecule in gene therapy-based approaches to treat difficult cancers.

Genetic mutations affecting mitochondrial fission and fusion proteins cause human neurological disorders, but are assumed to be well tolerated in yeast. The conserved mitochondrial fission protein Dnm1/Drp1 is required for normal mitochondrial division, but also promotes cell death in mammals and yeast. Fis1, an outer mitochondrial membrane-anchored receptor for Dnm1/Drp1, also can promote cell death in mammals, but appears to have prosurvival activity in yeast. Here we report that deletion of the FIS1 gene in yeast consistently results in acquisition of a secondary mutation that confers sensitivity to cell death. In several independently derived FIS1 knockouts, tiling arrays and genomic sequencing identified the secondary mutation as a premature termination in the same stress-response gene, WHI2. The WHI2 mutation rescues the mitochondrial respiratory defect (petite formation) caused by FIS1 deficiency, but also causes a failure to suppress cell growth during amino-acid deprivation. Thus, loss of Fis1 drives the selection for specific compensatory mutations that confer defective growth control and cell death regulation, characteristic of human tumor cells. The important long-term survival function of Fis1 that is compensated by WHI2 mutation appears to be independent of fission factor Dnm1/Drp1 and its adaptor Mdv1, but may be mediated through a second adaptor Caf4, as WHI2 is also mutated in a CAF4 knockout.

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation. Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation, the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L), in the regulation of erythroid maturation through mitochondrial autophagy. Nix(-/-) mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix(-/-) mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (DeltaPsi(m)), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of DeltaPsi(m) and restored the sequestration of mitochondria into autophagosomes in Nix(-/-) erythroid cells. These results suggest that Nix-dependent loss of DeltaPsi(m) is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.

Cytokines affect a variety of cellular functions, including regulation of cell numbers by suppression of programmed cell death. Suppression of apoptosis requires receptor signalling through the activation of Janus kinases and the subsequent regulation of members of the B-cell lymphoma 2 (Bcl-2) family. Here we demonstrate that a Bcl-2-family-related protein, Hax1, is required to suppress apoptosis in lymphocytes and neurons. Suppression requires the interaction of Hax1 with the mitochondrial proteases Parl (presenilin-associated, rhomboid-like) and HtrA2 (high-temperature-regulated A2, also known as Omi). These interactions allow Hax1 to present HtrA2 to Parl, and thereby facilitates the processing of HtrA2 to the active protease localized in the mitochondrial intermembrane space. In mouse lymphocytes, the presence of processed HtrA2 prevents the accumulation of mitochondrial-outer-membrane-associated activated Bax, an event that initiates apoptosis. Together, the results identify a previously unknown sequence of interactions involving a Bcl-2-family-related protein and mitochondrial proteases in the ability to resist the induction of apoptosis when cytokines are limiting.

p53-upregulated modulator of apoptosis (PUMA) is a BH3-only Bcl-2 family protein and an essential mediator of DNA damage-induced apoptosis. PUMA is localized in the mitochondria and induces apoptosis through the mitochondrial pathway. However, the mechanisms of PUMA-induced apoptosis remain unclear. In this study, we found that second mitochondria-derived activator of caspase (SMAC)/Diablo, a mitochondrial apoptogenic protein, mediates the proapoptotic function of PUMA by regulating PUMA-induced mitochondrial events. SMAC is consistently released into the cytosol in colon cancer cells undergoing PUMA-induced apoptosis. In SMAC-deficient cells, execution of PUMA-induced apoptosis is abrogated, in company with decreases in caspase activation, cytosolic release of cytochrome c and collapse of mitochondrial membrane potential. Reconstituting SMAC expression restored these events in the SMAC-deficient cells. Furthermore, SMAC and agents that mimic the inhibitor of apoptosis proteins (IAPs) inhibition function of SMAC significantly sensitize cells to PUMA-induced apoptosis. These results demonstrate an important role of SMAC in executing DNA damage-induced and PUMA-mediated apoptosis and suggest that SMAC participates in a feedback amplification loop to promote cytochrome c release and other mitochondrial events in apoptosis.

Deficiency of citrin due to mutations of the SLC25A13 gene causes adult-onset type II citrullinemia (CTLN2) and one type of neonatal intrahepatic cholestasis (NICCD). About half of the NICCD patients are detected based on high galactose, phenylalanine, and/or methionine concentrations on newborn mass screening (NMS). To clarify the perinatal and neonatal effects and the inconsistent results on NMS, we examined aminograms, the levels of bile acids and galactose in dried blood spots for NMS from 20 patients with NICCD. Birth weight was low for gestational age (-1.4 +/- 0.7 SD). Affected fetuses may have suffered intrauterine citrin deficiency. The first abnormality detected after birth was citrullinemia, and 19 of 20 patients had citrulline levels higher than +2 SD of controls. Tyrosine, phenylalanine, methionine, galactose, and bile acids were less affected than citrulline on d 5 after birth. Galactose and bile acids levels were increased at 1 mo in comparison with d 5 after birth due to impairment of the cytosolic NADH reducing-equivalent supply into mitochondria of hepatocytes. Patients with negative findings on NMS had low levels of total 20 amino acids. Citrulline/serine, citrulline /leucine plus isoleucine, and citrulline/total amino acids ratios, controlled for the confounding effect of low amount of total amino acids, were higher in all patients than +2 SD, +2 SD, and +3 SD of controls, respectively. NMS for citrin deficiency (frequency of homozygote with SLC25A13 mutation: 1/10,000-1/38,000 in East Asia) will be useful for clarification of the clinical course, treatment, and prevention of this disease.