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Ferdinando Palmieri - One of the best experts on this subject based on the ideXlab platform.
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Asymmetric dimethylarginine is transported by the Mitochondrial Carrier SLC25A2
Amino Acids, 2016Co-Authors: Vito Porcelli, Antonella Longo, Luigi Palmieri, Ellen I. Closs, Ferdinando PalmieriAbstract:Asymmetric dimethyl l -arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the Carrier protein(s) that transport ADMA across the inner Mitochondrial membrane. It was found that the recombinant, purified Mitochondrial solute Carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known Mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38 ± 0.04 mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the Mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function.
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antiporters of the Mitochondrial Carrier family
Current Topics in Membranes, 2014Co-Authors: Magnus Monne, Ferdinando PalmieriAbstract:The eukaryotic transport protein family SLC25 consists of Mitochondrial Carriers (MCs) that are recognized on the sequence level by a threefold repeated and conserved signature motif. The majority of MCs characterized so far catalyzes strict exchanges of substrates across the Mitochondrial inner membrane. The substrates are nucleotides, metabolic intermediates, and cofactors that are required in cytoplasmic and matrix metabolism. This review summarizes and discusses the current knowledge of the antiport mechanism(s) of MCs that has been deduced from determining transport characteristics and by analyzing structural, sequence, and mutagenesis data. The mode of transport varies among different MCs with respect to how the substrate translocation depends on the electrical and pH gradients across the Mitochondrial inner membrane, for example, the ADP/ATP Carrier is electrogenic (electrophoretic), the GTP/GDP Carrier is dependent on the pH gradient, the aspartate/glutamate Carrier is dependent on both, and the oxoglutarate/malate Carrier is independent of them. The structure of the bovine ADP/ATP Carrier consists of a six-transmembrane α-helix bundle with a pseudo-threefold symmetry and a closed matrix gate. By using this structure as a template in homology modeling, residues engaged in substrate binding and the formation of a cytoplasmic gate in MCs have been proposed. The functional importance of the residues of the binding site, the matrix, and the cytoplasmic gates is supported by transport activities of different MCs with single point mutations. Cumulative evidence has been used to postulate a general transport mechanism for MCs.
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Erratum: The Saccharomyces cerevisiae gene YPR011c encodes a Mitochondrial transporter of adenosine 5′-phosphosulfate and 3′-phospho-adenosine 5′-phosphosulfate (Biochimica et Biophysica Acta - Bioenergetics (2014) 326 (334))
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014Co-Authors: Simona Todisco, Maria Antonietta Di Noia, Alessandra Castegna, Francesco M. Lasorsa, Eleonora Paradies, Ferdinando PalmieriAbstract:The genome of Saccharomyces cerevisiae contains 35 members of the Mitochondrial Carrier family, nearly all of which have been functionally characterized. In this study, the identification of the Mitochondrial Carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this Carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of Mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated Mitochondrial transport of APS occurs in S. cerevisiae under thermal stress condition
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The Saccharomyces cerevisiae gene YPR011c encodes a Mitochondrial transporter of adenosine 5'-phosphosulfate and 3'-phospho-adenosine 5'-phosphosulfate.
Biochimica et biophysica acta, 2013Co-Authors: Simona Todisco, Maria Antonietta Di Noia, Alessandra Castegna, Francesco M. Lasorsa, Eleonora Paradies, Ferdinando PalmieriAbstract:The genome of Saccharomyces cerevisiae contains 35 members of the Mitochondrial Carrier family, nearly all of which have been functionally characterized. In this study, the identification of the Mitochondrial Carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this Carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of Mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated Mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.
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the human gene slc25a17 encodes a peroxisomal transporter of coenzyme a fad and nad
Biochemical Journal, 2012Co-Authors: Gennaro Agrimi, Pasquale Scarcia, Annamaria Russo, Ferdinando PalmieriAbstract: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.
Giuseppe Fiermonte - One of the best experts on this subject based on the ideXlab platform.
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the human uncoupling proteins 5 and 6 ucp5 slc25a14 and ucp6 slc25a30 transport sulfur oxyanions phosphate and dicarboxylates
Biochimica et Biophysica Acta, 2019Co-Authors: Ruggiero Gorgoglione, Vito Porcelli, Luigi Palmieri, Maria Antonietta Di Noia, Magnus Monne, Antonella Santoro, Lucia Daddabbo, Angelo Vozza, Giuseppe FiermonteAbstract:The human genome encodes 53 members of the solute Carrier family 25 (SLC25), also called the Mitochondrial Carrier family. In this work, two members of this family, UCP5 (BMCP1, brain Mitochondrial Carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney Mitochondrial Carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both Carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other Mitochondrial Carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H2S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H2S.
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MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria
Nature Cell Biology, 2010Co-Authors: Yehudit Zaltsman, Michal Schwarz, Giuseppe Fiermonte, Liat Shachnai, Natalie Yivgi-ohana, Maria Maryanovich, Riekelt H. Houtkooper, Frédéric Maxime Vaz, Francesco De Leonardis, Ferdinando PalmieriAbstract:The BH3-only BID protein (BH3-interacting domain death agonist) has a critical function in the death-receptor pathway in the liver by triggering Mitochondrial outer membrane permeabilization (MOMP). Here we show that MTCH2/MIMP (Mitochondrial Carrier homologue 2/Met-induced Mitochondrial protein), a novel truncated BID (tBID)-interacting protein, is a surface-exposed outer Mitochondrial membrane protein that facilitates the recruitment of tBID to mitochondria. Knockout of MTCH2/MIMP in embryonic stem cells and in mouse embryonic fibroblasts hinders the recruitment of tBID to mitochondria, the activation of Bax/Bak, MOMP, and apoptosis. Moreover, conditional knockout of MTCH2/MIMP in the liver decreases the sensitivity of mice to Fas-induced hepatocellular apoptosis and prevents the recruitment of tBID to liver mitochondria both in vivo and in vitro . In contrast, MTCH2/MIMP deletion had no effect on apoptosis induced by other pro-apoptotic Bcl-2 family members and no detectable effect on the outer membrane lipid composition. These loss-of-function models indicate that MTCH2/MIMP has a critical function in liver apoptosis by regulating the recruitment of tBID to mitochondria. BH3-only protein tBID induces Mitochondrial outer membrane permeabilization in response to death receptor activation. The Mitochondrial Carrier homologue 2 (MTCH2) protein acts as the tBID receptor on the mitochondria and is required for fas-induced cell death in mouse liver.
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molecular identification and functional characterization of arabidopsis thaliana Mitochondrial and chloroplastic nad Carrier proteins
Journal of Biological Chemistry, 2009Co-Authors: Ferdinando Palmieri, Joachim Tjaden, Gennaro Agrimi, Giuseppe Fiermonte, Benjamin Rieder, A Ventrella, Emanuela Blanco, Adriano Nunesnesi, A U Trauth, Simon KirchbergerAbstract:The Arabidopsis thaliana L. genome contains 58 membrane proteins belonging to the Mitochondrial Carrier family. Two Mitochondrial Carrier family members, here named AtNDT1 and AtNDT2, exhibit high structural similarities to the Mitochondrial nicotinamide adenine dinucleotide (NAD+) Carrier ScNDT1 from bakers' yeast. Expression of AtNDT1 or AtNDT2 restores Mitochondrial NAD+ transport activity in a yeast mutant lacking ScNDT. Localization studies with green fluorescent protein fusion proteins provided evidence that AtNDT1 resides in chloroplasts, whereas only AtNDT2 locates to mitochondria. Heterologous expression in Escherichia coli followed by purification, reconstitution in proteoliposomes, and uptake experiments revealed that both Carriers exhibit a submillimolar affinity for NAD+ and transport this compound in a counter-exchange mode. Among various substrates ADP and AMP are the most efficient counter-exchange substrates for NAD+. Atndt1- and Atndt2-promoter-GUS plants demonstrate that both genes are strongly expressed in developing tissues and in particular in highly metabolically active cells. The presence of both Carriers is discussed with respect to the subcellular localization of de novo NAD+ biosynthesis in plants and with respect to both the NAD+-dependent metabolic pathways and the redox balance of chloroplasts and mitochondria.
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a novel member of solute Carrier family 25 slc25a42 is a transporter of coenzyme a and adenosine 3 5 diphosphate in human mitochondria
Journal of Biological Chemistry, 2009Co-Authors: Giuseppe Fiermonte, Eleonora Paradies, Simona Todisco, Carlo M T Marobbio, Ferdinando PalmieriAbstract:Mitochondrial Carriers are a family of proteins that transport metabolites, nucleotides, and cofactors across the inner Mitochondrial membrane thereby connecting cytosolic and matrix functions. The essential cofactor coenzyme A (CoA) is synthesized outside the Mitochondrial matrix and therefore must be transported into mitochondria where it is required for a number of fundamental processes. In this work we have functionally identified and characterized SLC25A42, a novel human member of the Mitochondrial Carrier family. The SLC25A42 gene (Haitina, T., Lindblom, J., Renstrom, T., and Fredriksson, R., 2006, Genomics 88, 779–790) was overexpressed in Escherichia coli, purified, and reconstituted into phospholipid vesicles. Its transport properties, kinetic parameters, and targeting to mitochondria demonstrate that SLC25A42 protein is a Mitochondrial transporter for CoA and adenosine 3′,5′-diphosphate. SLC25A42 catalyzed only a counter-exchange transport, exhibited a high transport affinity for CoA, dephospho-CoA, ADP, and adenosine 3′,5′-diphosphate, was saturable and inhibited by bongkrekic acid and other inhibitors of Mitochondrial Carriers to various degrees. The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intraMitochondrial (deoxy)adenine nucleotides and adenosine 3′,5′-diphosphate. This is the first time that a Mitochondrial Carrier for CoA and adenosine 3′,5′-diphosphate has been characterized biochemically.
Magnus Monne - One of the best experts on this subject based on the ideXlab platform.
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the human uncoupling proteins 5 and 6 ucp5 slc25a14 and ucp6 slc25a30 transport sulfur oxyanions phosphate and dicarboxylates
Biochimica et Biophysica Acta, 2019Co-Authors: Ruggiero Gorgoglione, Vito Porcelli, Luigi Palmieri, Maria Antonietta Di Noia, Magnus Monne, Antonella Santoro, Lucia Daddabbo, Angelo Vozza, Giuseppe FiermonteAbstract:The human genome encodes 53 members of the solute Carrier family 25 (SLC25), also called the Mitochondrial Carrier family. In this work, two members of this family, UCP5 (BMCP1, brain Mitochondrial Carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney Mitochondrial Carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both Carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other Mitochondrial Carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H2S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H2S.
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antiporters of the Mitochondrial Carrier family
Current Topics in Membranes, 2014Co-Authors: Magnus Monne, Ferdinando PalmieriAbstract:The eukaryotic transport protein family SLC25 consists of Mitochondrial Carriers (MCs) that are recognized on the sequence level by a threefold repeated and conserved signature motif. The majority of MCs characterized so far catalyzes strict exchanges of substrates across the Mitochondrial inner membrane. The substrates are nucleotides, metabolic intermediates, and cofactors that are required in cytoplasmic and matrix metabolism. This review summarizes and discusses the current knowledge of the antiport mechanism(s) of MCs that has been deduced from determining transport characteristics and by analyzing structural, sequence, and mutagenesis data. The mode of transport varies among different MCs with respect to how the substrate translocation depends on the electrical and pH gradients across the Mitochondrial inner membrane, for example, the ADP/ATP Carrier is electrogenic (electrophoretic), the GTP/GDP Carrier is dependent on the pH gradient, the aspartate/glutamate Carrier is dependent on both, and the oxoglutarate/malate Carrier is independent of them. The structure of the bovine ADP/ATP Carrier consists of a six-transmembrane α-helix bundle with a pseudo-threefold symmetry and a closed matrix gate. By using this structure as a template in homology modeling, residues engaged in substrate binding and the formation of a cytoplasmic gate in MCs have been proposed. The functional importance of the residues of the binding site, the matrix, and the cytoplasmic gates is supported by transport activities of different MCs with single point mutations. Cumulative evidence has been used to postulate a general transport mechanism for MCs.
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the mimivirus genome encodes a Mitochondrial Carrier that transports datp and dttp
Journal of Virology, 2007Co-Authors: Magnus Monne, Alan J Robinson, Christoph Boes, Michael E Harbour, Ian M Fearnley, Edmund R S KunjiAbstract:Members of the Mitochondrial Carrier family have been reported in eukaryotes only, where they transport metabolites and cofactors across the Mitochondrial inner membrane to link the metabolic pathways of the cytosol and the matrix. The genome of the giant virus Mimiviridae mimivirus encodes a member of the Mitochondrial Carrier family of transport proteins. This viral protein has been expressed in Lactococcus lactis and is shown to transport dATP and dTTP. As the 1.2-Mb double-stranded DNA mimivirus genome is rich in A and T residues, we speculate that the virus is using this protein to target the host mitochondria as a source of deoxynucleotides for its replication.
Martin D. Brand - One of the best experts on this subject based on the ideXlab platform.
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Not all Mitochondrial Carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1
Bioscience Reports, 2009Co-Authors: Paul G Crichton, Nadeene Parker, Antonio J Vidal-puig, Martin D. BrandAbstract:The Mitochondrial permeability transition pore (mPTP) is a non-specific channel that forms in the Mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be comprised of the adenine nucleotide translocase (ANT), voltage dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the Mitochondrial Carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility we have studied the permeability transition properties of brown adipose tissue mitochondria in which levels of the Mitochondrial Carrier protein, uncoupling protein 1 (UCP1), can exceed those of ANT. Using an improved spectroscopic assay we have quantified mPTP formation in de-energised mitochondria from wild type and Ucp1 knockout mice and assessed the dependence of pore formation on UCP1. When correctly normalised for differences in Mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (~50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (~80% inhibited, compared to ~60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in brown adipose tissue and that a role in mPTP formation is not a general characteristic of the Mitochondrial Carrier protein family but is more likely to be restricted to specific members including ANT.
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Not all Mitochondrial Carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1
Bioscience reports, 2009Co-Authors: Paul G Crichton, Nadeene Parker, Antonio J Vidal-puig, Martin D. BrandAbstract:The mPTP (Mitochondrial permeability transition pore) is a non-specific channel that is formed in the Mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the Mitochondrial Carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the Mitochondrial Carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in Mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the Mitochondrial Carrier protein family but is, more likely, restricted to specific members including ANT.
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Does Any Yeast Mitochondrial Carrier Have a Native Uncoupling Protein Function
Journal of bioenergetics and biomembranes, 2002Co-Authors: Damien Roussel, Marilyn Harding, Michael J. Runswick, John E. Walker, Martin D. BrandAbstract:In this study, we explore the hypothesis that some member of the Mitochondrial Carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria. Twenty-seven of the 35 yeast Mitochondrial Carrier genes were independently disrupted in Saccharomyces cerevisiae. Six knockout strains did not grow on nonfermentable carbon sources such as lactate. Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured. None of the 21 Carriers contributed significantly to the basal proton leak of yeast mitochondria. A possible exception was the succinate/fumarate Carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in Mitochondrial function. If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown.
Luigi Palmieri - One of the best experts on this subject based on the ideXlab platform.
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the human uncoupling proteins 5 and 6 ucp5 slc25a14 and ucp6 slc25a30 transport sulfur oxyanions phosphate and dicarboxylates
Biochimica et Biophysica Acta, 2019Co-Authors: Ruggiero Gorgoglione, Vito Porcelli, Luigi Palmieri, Maria Antonietta Di Noia, Magnus Monne, Antonella Santoro, Lucia Daddabbo, Angelo Vozza, Giuseppe FiermonteAbstract:The human genome encodes 53 members of the solute Carrier family 25 (SLC25), also called the Mitochondrial Carrier family. In this work, two members of this family, UCP5 (BMCP1, brain Mitochondrial Carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney Mitochondrial Carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both Carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other Mitochondrial Carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H2S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H2S.
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Asymmetric dimethylarginine is transported by the Mitochondrial Carrier SLC25A2
Amino Acids, 2016Co-Authors: Vito Porcelli, Antonella Longo, Luigi Palmieri, Ellen I. Closs, Ferdinando PalmieriAbstract:Asymmetric dimethyl l -arginine (ADMA) is generated within cells and in mitochondria when proteins with dimethylated arginine residues are degraded. The aim of this study was to identify the Carrier protein(s) that transport ADMA across the inner Mitochondrial membrane. It was found that the recombinant, purified Mitochondrial solute Carrier SLC25A2 when reconstituted into liposomes efficiently transports ADMA in addition to its known substrates arginine, lysine, and ornithine and in contrast to the other known Mitochondrial amino acid transporters SLC25A12, SLC25A13, SLC25A15, SLC25A18, SLC25A22, and SLC25A29. The widely expressed SLC25A2 transported ADMA across the liposomal membrane in both directions by both unidirectional transport and exchange against arginine or lysine. The SLC25A2-mediated ADMA transport followed first-order kinetics, was nearly as fast as the transport of the best SLC25A2 substrates known so far, and was highly specific as symmetric dimethylarginine (SDMA) was not transported at all. Furthermore, ADMA inhibited SLC25A2 activity with an inhibition constant of 0.38 ± 0.04 mM, whereas SDMA inhibited it poorly. We propose that a major function of SLC25A2 is to export ADMA from mitochondria missing the Mitochondrial ADMA-metabolizing enzyme AGXT2. There is evidence that ADMA can also be imported into mitochondria, e.g., in kidney proximal tubulus cells, to be metabolized by AGXT2. SLC25A2 may also mediate this transport function.
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Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae
The Journal of biological chemistry, 2010Co-Authors: Alessandra Castegna, Luigi Palmieri, Gennaro Agrimi, Pasquale Scarcia, Hanspeter Rottensteiner, Iolanda Spera, Lucrezia Germinario, Ferdinando PalmieriAbstract: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 ΔYHM2 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 ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.
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Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in
2010Co-Authors: Alessandra Castegna, Luigi Palmieri, Gennaro Agrimi, Pasquale Scarcia, Hanspeter Rottensteiner, Iolanda Spera, Lucrezia Germinario, Ferdinando PalmieriAbstract: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 sulfhydrylblocking 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 YHM2 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 theYHM2 strain and more so by theYHM2ZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.
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molecular identification of three arabidopsis thaliana Mitochondrial dicarboxylate Carrier isoforms organ distribution bacterial expression reconstitution into liposomes and functional characterization
Biochemical Journal, 2008Co-Authors: Luigi Palmieri, Ferdinando Palmieri, Nathalie Picault, Roberto Arrigoni, Evelyne Besin, Michael HodgesAbstract:Screening of the Arabidopsis thaliana genome revealed three potential homologues of mammalian and yeast Mitochondrial DICs (dicarboxylate Carriers) designated as DIC1, DIC2 and DIC3, each belonging to the Mitochondrial Carrier protein family. DIC1 and DIC2 are broadly expressed at comparable levels in all the tissues investigated. DIC1–DIC3 have been reported previously as uncoupling proteins, but direct transport assays with recombinant and reconstituted DIC proteins clearly demonstrate that their substrate specificity is unique to plants, showing the combined characteristics of the DIC and oxaloacetate Carrier in yeast. Indeed, the Arabidopsis DICs transported a wide range of dicarboxylic acids including malate, oxaloacetate and succinate as well as phosphate, sulfate and thiosulfate at high rates, whereas 2-oxoglutarate was revealed to be a very poor substrate. The role of these plant Mitochondrial DICs is discussed with respect to other known Mitochondrial Carrier family members including uncoupling proteins. It is proposed that plant DICs constitute the membrane component of several metabolic processes including the malate–oxaloacetate shuttle, the most important redox connection between the mitochondria and the cytosol.