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Mitochondrial RTG-dependent retrograde signaling, whose regulators have been characterized in Saccharomyces cerevisiae, plays a recognized role under various environmental stresses. Of special significance, the activity of the transcriptional complex Rtg1/3 has been shown to be modulated by Hog1, the master regulator of the high osmolarity glycerol pathway, in response to osmotic stress. The present work focuses on the role of RTG signaling in salt-induced osmotic stress and its interaction with HOG1. Wild-type and mutant cells, lacking HOG1 and/or RTG genes, are compared with respect to cell growth features, retrograde signaling activation and mitochondrial function in the presence and in the absence of high osmostress. We show that RTG2, the main upstream regulator of the RTG pathway, contributes to osmoadaptation in an HOG1-dependent manner and that, with RTG3, it is notably involved in a late phase of growth. Our data demonstrate that impairment of RTG signaling causes a decrease in mitochondrial respiratory capacity exclusively under osmostress. Overall, these results suggest that HOG1 and the RTG pathway may interact sequentially in the stress signaling cascade and that the RTG pathway may play a role in inter-organellar metabolic communication for osmoadaptation.

Mitochondrial citrate carrier plays a central role in exporting acetyl-CoA in the form of citrate from mitochondria to cytosol thereby connecting carbohydrate catabolism and lipogenesis. In this study, Yarrowia lipolytica mitochondrial citrate carrier was functionally defined and characterized. Firstly, deletion of Y. lipolytica YlCTP1 and YlYHM2 genes coding putative tricarboxylate mitochondrial carriers were performed. ΔYlctp1 strain did not differ significantly from wild type strain in terms of growth rate, organic acids and lipid production. In contrast, ΔYlyhm2 strain did not grow in liquid citrate-containing minimal medium. Moreover, in glucose-containing lipogenic medium YlYHM2 null mutant strain did not produce citric acid; the production of isocitric acid and lipids were decreased. Reintroduction of YlYHM2 gene as well as heterologous expression of Aspergillus niger gene AnYHM2 into ΔYlyhm2 strain restored the growth in minimal citrate medium and even enhanced citric acid production by 45% in both variants compared with wild type strain during test tube cultivation. Mitochondrial extracts isolated from YlYHM2 null mutant and wild type strain were incorporated into liposomes; citrate/citrate and α-ketoglutarate/α-ketoglutarate homoexchange activities were reduced by 87% and 40% in ΔYlyhm2 strain, respectively, compared with the wild type, whereas citratein/α-ketoglutarateout and α-ketoglutaratein/citrateout heteroexchanges were decreased by 87% and 95%, respectively. YlYhm2p was expressed in Escherichia coli, purified and reconstituted into liposomes. Besides high efficiency to citrate and α-ketoglutarate transport, YlYhm2p also transported oxaloacetate, succinate, fumarate, and to a much lesser extent, aconitate, malate, isocitrate, oxoadipate, and glutamate. The activity of reconstituted YlYhm2p was inhibited strongly by SH-blocking reagents, pyridoxal-5′-phosphate, and partly by N-ethylmaleimide. Co-expression of YlYHM2 and adenosine monophosphate deaminase YlAMPD genes resulted in the production of 49.7 g/L of citric acid during test tube cultivation, whereas wild type strain accumulated 30.1 g/L of citric acid. Large-scale cultivation in bioreactor of the engineered strain resulted in 97.1 g/L of citric acid production with a process selectivity of 94.2% and an overall citric acid yield of 0.5 g/g. The maximal specific rate of citric acid synthesis was 0.93 g/L/h. Therefore, the physiological role of YlYhm2p in glucose-containing medium is to catalyze both import of citrate into mitochondria for catabolic reactions and export of citrate as a source of acetyl-CoA from mitochondria. Possible shuttles for citrate exporting are discussed. Moreover, for the first time evidence has been given for the improvement of TCA cycle intermediate production by manipulation of a gene coding a mitochondrial carrier.

During cultivation under nitrogen starvation, Yarrowia lipolytica produces a mixture of citric acid and isocitric acid whose ratio is mainly determined by the carbon source used. We report that mitochondrial succinate–fumarate carrier YlSfc1 controls isocitric acid efflux from mitochondria. YlSfc1 purified and reconstituted into liposomes transports succinate, fumarate, oxaloacetate, isocitrate and α-ketoglutarate. YlSFC1 overexpression determined the inversion of isocitric acid/citric acid ratio towards isocitric acid, resulting in 33.4 ± 1.9 g/L and 43.3 ± 2.8 g/L of ICA production in test-tube cultivation with glucose and glycerol, respectively. These titers represent a 4.0 and 6.3-fold increase compared to the wild type. YlSFC1 gene expression was repressed in the wild type strain grown in glucose-based medium compared to olive oil medium explaining the reason for the preferred citric acid production during Y. lipolytica growth on carbohydrates. Coexpression of YlSFC1 and adenosine monophosphate deaminase YlAMPD genes together with inactivation of citrate mitochondrial carrier YlYHM2 gene enhanced isocitric acid accumulation up to 41.4 ± 4.1 g/L with an isocitric acid/citric acid ratio of 14.3 in a small-scale cultivation with glucose as a carbon source. During large-scale cultivation with glucose pulse-feeding, the engineered strain produced 136.7 ± 2.5 g/L of ICA with a process selectivity of 88.1%, the highest reported titer and selectivity to date. These results represent the first reported isocitric acid secretion by Y. lipolytica as a main organic acid during cultivation on carbohydrate. Moreover, we demonstrate for the first time that the replacement of one mitochondrial transport system for another can be an efficient tool for switching product accumulation.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour. UCP2 is shown in yeast and mammalian cells to transport aspartate out of mitochondria, thus enabling KRAS-mutated pancreatic ductal adenocarcinoma cells to perform glutaminolysis to support cancer growth.

In Saccharomyces cerevisiae the export of 2-oxoglutarate from the mitochondria, catalyzed by Yhm2p, Odc1p and Odc2p or by at least one of these transporters, has recently been shown to be essential for glutamate biosynthesis in glucose-supplemented minimal synthetic (SM) medium without glutamate, because the triple mutant yhm2∆odc1∆odc2∆ displays a growth defect under these conditions. Surprisingly, in this study it was found that yhm2∆odc1∆odc2∆ cells grow like wild-type (WT) cells in the same medium supplemented with non-fermentable carbon sources. Direct transport assays of 2-oxoglutarate/2-oxoglutarate homoexchange activity in mitochondria from WT and yhm2∆odc1∆odc2∆ cells (solubilized and reconstituted into liposomes) showed that the mitochondrial extract from yhm2∆odc1∆odc2∆ was completely inactive at variance with that from WT cells, showing that S. cerevisiae mitochondria do not contain additional proteins capable of catalyzing 2-oxoglutarate transport efficiently besides Yhm2p, Odc1p and Odc2p. Furthermore, quantitative real-time PCR experiments showed that in both WT and yhm2∆odc1∆odc2∆ cells the expression of GDH1 is low on lactate and high on glucose and, vice versa, the expression of GDH3 is high on lactate and low on glucose. These results may be interpreted to indicate that in S. cerevisiae, grown in glucose-supplemented SM medium, glutamate is synthesized by Gdh1p in the cytosol, whereas in lactate-supplemented SM medium glutamate is synthesized by Gdh3p in the mitochondria; therefore, the pathway of ammonia assimilation under fermentative conditions requires export of 2-oxoglutarate from the mitochondria, whereas the alternative pathway under respiratory conditions does not.

Neuromuscular diseases (NMDs) are dysfunctions that involve skeletal muscle and cause incorrect communication between the nerves and muscles. The specific causes of NMDs are not well known, but most of them are caused by genetic mutations. NMDs are generally progressive and entail muscle weakness and fatigue. Muscular impairments can differ in onset, severity, prognosis, and phenotype. A multitude of possible injury sites can make diagnosis of NMDs difficult. Mitochondria are crucial for cellular homeostasis and are involved in various metabolic pathways; for this reason, their dysfunction can lead to the development of different pathologies, including NMDs. Most NMDs due to mitochondrial dysfunction have been associated with mutations of genes involved in mitochondrial biogenesis and metabolism. This review is focused on some mitochondrial routes such as the TCA cycle, OXPHOS, and β-oxidation, recently found to be altered in NMDs. Particular attention is given to the alterations found in some genes encoding mitochondrial carriers, proteins of the inner mitochondrial membrane able to exchange metabolites between mitochondria and the cytosol. Briefly, we discuss possible strategies used to diagnose NMDs and therapies able to promote patient outcome.

Itaconic acid (IA) is a naturally occurring dicarboxylic acid with applications in the manufacture of polymers. IA can be produced by fermentation using the fungi Aspergillus terreus or Ustilago maydis as biocatalysts. Indirect evidence has suggested that the mitochondrial carriers U. maydis Um_Mtt1 and A. terreus At_MttA export mitochondrially synthesized cis-aconitate to the cytosol for IA synthesis using malate as a countersubstrate. Here, by assaying the transport features of recombinant Um_Mtt1 and At_MttA in reconstituted liposomes, we find that both proteins efficiently transport cis-aconitate, but malate is well transported only by Um_Mtt1 and 2-oxoglutarate only by At_MttA. Bioinformatic analysis shows that Um_Mtt1 and At_MttA form a distinctive mitochondrial carrier subfamily. Our data show that although fulfilling the same physiological task, Um_Mtt1 and At_MttA have different biochemical features.

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N1-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N1-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N1-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N1-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N1-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype–genotype correlation studies. Electronic supplementary material The online version of this article (10.1007/s10545-017-0106-7) contains supplementary material, which is available to authorized users.

The nuclear genes of Saccharomyces cerevisiae YHM2, ODC1 and ODC2 encode three transporters that are localized in the inner mitochondrial membrane. In this study, the roles of YHM2, ODC1 and ODC2 in the assimilation of nitrogen and in the biosynthesis of lysine have been investigated. Both the odc1Δodc2Δ double knockout and the yhm2Δ mutant grew similarly as the YPH499 wild-type strain on synthetic minimal medium (SM) containing 2% glucose and ammonia as the main nitrogen source. In contrast, the yhm2Δodc1Δodc2Δ triple knockout exhibited a marked growth defect under the same conditions. This defect was fully restored by the individual expression of YHM2, ODC1 or ODC2 in the triple deletion strain. Furthermore, the lack of growth of yhm2Δodc1Δodc2Δ on 2% glucose SM was rescued by the addition of glutamate, but not glutamine, to the medium. Using lysine-prototroph YPH499-derived strains, the yhm2Δodc1Δodc2Δ knockout (but not the odc1Δodc2Δ and yhm2Δ mutants) also displayed a growth defect in lysine biosynthesis on 2% glucose SM, which was rescued by the addition of lysine and, to a lesser extent, by the addition of 2-aminoadipate. Additional analysis of the triple mutant showed that it is not respiratory-deficient and does not display mitochondrial DNA instability. These results provide evidence that only the simultaneous absence of YHM2, ODC1 and ODC2 impairs the export from the mitochondrial matrix of i) 2-oxoglutarate which is necessary for the synthesis of glutamate and ammonium fixation in the cytosol and ii) 2-oxoadipate which is required for lysine biosynthesis in the cytosol. Finally, the data presented allow one to suggest that the yhm2Δodc1Δodc2Δ triple knockout is suitable in complementation studies aimed at assessing the pathogenic potential of human SLC25A21 (ODC) mutations.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

BACKGROUND AND OBJECTIVE: Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia. METHODS: We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development. RESULTS: We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype. CONCLUSIONS: Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.

The mitochondrial citrate carrier (CIC) encoded by the SLC25A1 gene, catalyzes the export of citrate from mitochondria to the cytosol where it is broken into into acetyl-CoA and oxaloacetate. By exome sequencing we identified for the first time two pathogenic SLC25A1 variants in a patient that suffered from a severe neurodevelopmental syndrome. More recently we reported a novel homozygous mutation in the SLC25A1 gene in an affected sib pair. Both patients presented with myasthenia and impaired neuromuscular junction (NMJ) transmission whilst no neurodevelopment disorder has been observed [1]. Functional analysis of the pathogenic mutations has been performed upon reconstitution of recombinant proteins into liposomes. Interestingly, the newly identified mutation caused a milder activity impairment than the previously reported mutations suggesting a fundamental role of CIC in NM transmission whose defect was previously masked by the harsher phenotype. Knocking down the SLC25A1 orthologues in zebrafish embryos we observed abnormalities in the NMJ with an erratic outgrowth of neuronal axons toward muscle fiber. Using the CRISPR/CAS9 approach we obtained stable lines of C. elegans knocked-out in the SLC25A1 ortholog that showed partial resistance to Aldicarb, a specific inhibitor of cholinesterase activity that causes the paralysis of the worms. Altogether our data point to a key role of CIC in NMJ transmission most likely due to the requirement of acetyl-CoA supply for acetylcholine synthesis at the pre-synaptic nerve terminal.

The essential cofactors CoA, FAD and NAD+ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In the present study we have functionally identified and characterized SLC25A17 (solute carrier family 25 member 17), which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN and AMP, and to a lesser extent of NAD+, PAP (adenosine 3′,5′-diphosphate) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and was inhibited by pyridoxal 5′-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all of the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. The present paper is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.

The mitochondrial ADP/ATP carriers (AACs) catalyze the exchange of cytosolic ADP for matrix ATP. We have identified and characterized a novel member of the AAC subfamily of mitochondrial metabolite transport proteins, termed AAC4. The AAC4 gene maps to human chromosome 4q28.1, and its product AAC4 is 66–68% identical to human AAC 1–3 and is localized to mitochondria. AAC4 transcripts are exclusively present in liver, testis and brain unlike those of AAC 1–3. Consistent with its belonging to the AAC subfamily, upon heterologous expression and reconstitution into liposomes AAC4 exchanges ADP for ATP by an electrogenic antiport mechanism with high specificity and high sensitivity to carboxyatractyloside and bongkrekic acid.

Autosomal dominant and recessive forms of progressive external ophthalmoplegia (adPEO and arPEO) are mitochondrial disorders characterized by the presence of multiple deletions of mitochondrial DNA in affected tissues. Four adPEO-associated missense mutations have been identified in the ANT1 gene. In order to investigate their functional consequences on cellular physiology, we introduced three of them at equivalent positions in AAC2, the yeast orthologue of human ANT1. We demonstrate here that expression of the equivalent mutations in aac2-defective haploid strains of Saccharomyces cerevisiae results in (a) a marked growth defect on non-fermentable carbon sources, and (b) a concurrent reduction of the amount of mitochondrial cytochromes, cytochrome c oxidase activity and cellular respiration. The efficiency of ATP and ADP transport was variably affected by the different AAC2 mutations. However, irrespective of the absolute level of activity, the AAC2 pathogenic mutants showed a significant defect in ADP versus ATP transport compared with wild-type AAC2. In order to study whether a dominant phenotype, as in humans, could be observed, the aac2 mutant alleles were also inserted in combination with the endogenous wild-type AAC2 gene. The heteroallelic strains behaved as recessive for oxidative growth and petite-negative phenotype. In contrast, reduction in cytochrome content and increased mtDNA instability appeared to behave as dominant traits in heteroallelic strains. Our results indicate that S. cerevisiae is a suitable in vivo model to study the pathogenicity of the human ANT1 mutations and the pathophysiology leading to impairment of oxidative phosphorylation and damage of mtDNA integrity, as found in adPEO.

A GENiE (EU COST action, www.worm-genie.eu) workshop was held at the Babraham Institute in Cambridge, UK on April 19 and 20, 2017, to discuss global challenges around Caenorhabditis elegans metabol...

Background Agenesis of corpus callosum has been associated with several defects of the mitochondrial respiratory chain and the citric acid cycle. We now report the results of the biochemical and molecular studies of a patient with severe neurodevelopmental disease manifesting by agenesis of corpus callosum and optic nerve hypoplasia. Methods and results A mitochondrial disease was suspected in this patient based on the prominent excretion of 2-hydroxyglutaric acid and Krebs cycle intermediates in urine and the finding of increased reactive oxygen species content and decreased mitochondrial membrane potential in her fibroblasts. Whole exome sequencing disclosed compound heterozygosity for two pathogenic variants in the SLC25A1 gene, encoding the mitochondrial citrate transporter. These variants, G130D and R282H, segregated in the family and were extremely rare in controls. The mutated residues were highly conserved throughout evolution and in silico modeling investigations indicated that the mutations would have a deleterious effect on protein function, affecting either substrate binding to the transporter or its translocation mechanism. These predictions were validated by the observation that a yeast strain harbouring the mutations at equivalent positions in the orthologous protein exhibited a growth defect under stress conditions and by the loss of activity of citrate transport by the mutated proteins reconstituted into liposomes. Conclusions We report for the first time a patient with a mitochondrial citrate carrier deficiency. Our data support a role for citric acid cycle defects in agenesis of corpus callosum as already reported in patients with aconitase or fumarate hydratase deficiency.

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP(+) and GSH/GSSG ratios in the cytosol of DeltaYHM2 cells as well as an increase in the NADPH/NADP(+) ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the DeltaYHM2 strain and more so by the DeltaYHM2DeltaZWF1 strain upon H(2)O(2) exposure, implying that Yhm2p has an antioxidant function.

Ca2+ concentration in peroxisomal matrix ([Ca2+](perox)) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50-100 microm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+](perox) being approximately 20-fold higher than [Ca2+] in the cytosol ([Ca2+](cyt)). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+](perox) of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1 ) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no opht hal mopl egi a. A muscl e bi opsy showed t he presence of numerous ragged- red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a ΔpH across the peroxisomal membrane, acidic inside.

The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven. Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family. Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible. Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion. Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source. Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated. Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter. The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids.

Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Mitochondria play a central role in coupling glucose metabolism to insulin secretion. However, little is known on mitochondrial responses to specific stresses; i.e. low versus high glucose, saturated versus unsaturated fatty acids, or oxidative stress. INS-1E cells were exposed for 3 days to 5.6 mM glucose, 25 mM glucose, 0.4 mM palmitate, and 0.4 mM oleate. Culture at standard 11.1 mM glucose served as no-stress control and transient oxidative stress (200 µM H2O2 for 10 min at day 0) served as positive stressful condition. Mito-array analyzed transcripts of 60 mitochondrion-associated genes with special focus on members of the Slc25 family. Transcripts of interest were evaluated at the protein level by immunoblotting. Bioinformatics analyzed the expression profiles to delineate comprehensive networks. Chronic exposure to the different metabolic stresses impaired glucose-stimulated insulin secretion; revealing glucotoxicity and lipo-dysfunction. Both saturated and unsaturated fatty acids increased expression of the carnitine/acylcarnitine carrier CAC, whereas the citrate carrier CIC and energy sensor SIRT1 were specifically upregulated by palmitate and oleate, respectively. High glucose upregulated CIC, the dicarboxylate carrier DIC and glutamate carrier GC1. Conversely, it reduced expression of energy sensors (AMPK, SIRT1, SIRT4), metabolic genes, transcription factor PDX1, and anti-apoptotic Bcl2. This was associated with caspase-3 cleavage and cell death. Expression levels of GC1 and SIRT4 exhibited positive and negative glucose dose-response, respectively. Expression profiles of energy sensors and mitochondrial carriers were selectively modified by the different conditions, exhibiting stress-specific signatures.

Introduction Une exposition chronique des cellules pancreatiques beta a des stresses aussi varies qu’une concentration elevee de glucose, d’acides gras ou d’oxydants induit un dysfonctionnement du couplage entre le metabolisme du glucose et la secretion d’insuline, generalement lie au metabolisme mitochondrial et entraine l’apoptose. Afin de determiner les mecanismes moleculaires respectifs de ces differents stresses associes au diabete, nous avons voulu identifier de nouvelles cibles en combinant une analyse selective de l’expression de genes impliques dans la fonction mitochondriale avec des outils bio-informatiques recemment developpes. Un interet particulier a ete porte sur l’expression des transporteurs mitochondriaux de la famille Slc25a. Materiels et methodes Les cellules INS-1E ont ete cultivees pendant trois jours en presence de stresses varies : concentrations de glucose basse (5,5 mM), intermediaire (11,6 mM) et elevee (25 mM), de 0,4 mM palmitate ou d’oleate ou a un stress oxydatif (200microM H2O2) transitoire de 10 minutes. Apres isolation de l’ARN total et synthese de l’ADNc, le profil d’expression des 57 genes d’interet selectionnes a ete obtenu par RT-PCR au moyen d’une carte micro-fluide (Mito-array). Les resultats ont ete integres dans le programme bio-informatique Cytoscape, afin d’etablir un reseau comprehensible des interactions geniques et de visualiser les changements complexes d’expression d’ARNm suite aux differents stresses. Resultats Parmi les 49 genes detectes dans les cellules INS-1E, nous avons identifie l’expression de 22 transporteurs mitochondriaux de la famille Slc25a. Une concentration elevee de glucose induit l’expression de plusieurs transporteurs (Slc25a1, Slc25a10, Slc25a13) ; resultats compatibles avec une activite anaplerotique/cataplerotique elevee. Le degre d’insaturation des acides gras modifie l’expression des nombreux genes de maniere opposee, suggerant des effets specifiques sur la cellule beta tels que toxicite versus dysfonctionnement. Conclusion Une meilleure comprehension des reseaux metaboliques alteres dans le dysfonctionnement des cellules beta suite a differentes stresses sera utile dans l’approche therapeutique du diabete.