Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster

none13noCoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.Vozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, GiuseppeVozza, Angelo; Leonardis, Francesco De; Paradies, Eleonora; Grassi, Anna De; Pierri, Ciro Leonardo; Parisi, Giovanni; Marobbio, Carlo Marya Thomas; Lasorsa, Francesco Massimo; Muto, Luigina; Capobianco, Loredana; Dolce, Vincenza; Raho, Susanna; Fiermonte, Giusepp

Diazossido per il trattamento dell'atassia di Friedreich

La presente invenzione ha per oggetto una preparazione farmaceutica per il trattamento dell’atassia di Friedreich (mancanza di coordinazione motoria) e per il trattamento o prevenzione delle patologie ad essa correlate. Conseguentemente si è messo a punto un procedimento per valutare i livelli di espressione della fratassina (proteina che nei soggetti affetti dalla patologia menzionata è estremamente ridotto)

Brevetto: Diazossido per il trattamento dell'atassia di Friedreich

La presente invenzione ha per oggetto una preparazione farmaceutica per il trattamento dell’atassia di Friedreich e per il trattamento o prevenzione delle patologie ad essa correlate. In particolare la presente invenzione ha per oggetto l’uso di diazossido o 7-cloro-3-metil-4H-1,2,4 benzotiazidina 1,1-diossido, in combinazione con glucosio e/o leucina, per il trattamento dell’atassia di Friedreich (FRDA) e per il trattamento o prevenzione delle patologie ad essa correlate

Diazoxide for the treatment of Friedreich's Ataxia

A pharmaceutical preparation treats Friedreich's ataxia and treats or prevents pathologies related thereto. In particular, the pharmaceutical preparation concerns the use of diazoxide or 7-chloro-3-methyl-4H-1,2,4 benzothiadiazine 1,1-dioxide, in combination with glucose and/or leucine, for the treatment of Friedreich's ataxia (FRDA) and for the treatment or prevention of pathologies related theret

alpha-Isopropylmalate, a leucine biosynthesis intermediate in yeast, is transported by the mitochondrial oxalacetate carrier

In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate

Rapamycin reduces oxidative stress in frataxin-deficient yeast cells

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria

Unidirectional transport of the mitochondrial GTP/GDP carrier in Saccharomyces cerevisiae

Ggc1p is a yeast mitochondrial carrier protein involved in the GTP/GDP transport. This protein encoded by YDL198c gene has been shown to be a multicopy suppressor (by an unknown mechanism) of the ability of the abf2 null mutant to grow at 37°C on glycerol. The ABF2 gene whose product is involved in mitochondrial genome maintenance in S. cerevisiae. Abf2Δ cells loose mtDNA at a high rate when grown in glucose medium and show a temperature-sensitive defect on non-fermentable carbon sources. The physiological role of Ggc1p in S. cerevisiae is the GTP transport into mitochondria, in exchange for intramitochondrially generated GDP. In addition, ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in their mitochondria; they are unable to grow on nonfermentable substrates, and they loose mtDNA. In the mitochondrial matrix, GTP is required as an energy source for protein synthesis; as a substrate for the synthesis of tRNA, mRNA, rRNA, and RNA primers; and as a phosphate group donor for the activity of GTP-AMP phosphotransferase and G proteins. In several organisms, GTP is synthesized in the mitochondria by succinyl-CoA ligase, which catalyzes the conversion of succinyl-CoA to succinate with the generation of GTP, and by nucleoside diphosphate kinase, which catalyzes the transfer of the phosphate from ATP to a nucleoside diphosphate, to yield nucleotide triphosphates. In S. cerevisiae, however, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space and it is absent in the matrix. These observations imply that in S. cerevisiae GTP has to be imported into the mitochondria probably via a carrier system embedded in the inner mitochondrial membrane. Here, this protein has been overexpressed in E. coli, reconstituted into phospholipid vesicles, and tested for a variety of potential substrates. When citrate is present, the carrier changes the transport activity, from an antiport mechanism to an uniport mechanism. A similar response has also been observed for the protein in the mitochondria. We conclude that uniport transport of GTP is involved in the homeostasis of guanine nucleotide pool in the mitochondrial matrix

TRANSPORT OF COENZYME A AND ADENOSINE 3'-5'DIPHOSPHATE ACROSS THE INNER MITOCHONDRIAL MEMBRANE: ROLE OF THE HUMAN SLC25A42 GENE PRODUCT

Mitochondrial carriers are a superfamily of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites, nucleotides and cofactors through this membrane, and thereby connect and/or regulate cytoplasm and matrix functions. Although several members of this family have been characterized, the functions of many others still remains unknown. For instance, the carrier responsible for coenzyme A (COA) transport into mitochondria has not yet been identified. CoA is an essential cytosolic-synthesized cofactor required in many intra-mitochondrial pathways, such as Krebs cycle, fatty acid β-oxidation and fatty acid synthesis (for the activation of mitochondrial ACP). In this work, we have identified SLC25A42 as the human gene responsible for the transport of CoA into mitochondria. The protein encoded by SLC25A42 is localized in the inner mitochondrial membrane and is ubiquitously expressed, aIthough at different levels. Its functional characterization has been carried out on bacterially expressed protein, upon its purification and reconstitution into phospholipid vesicles. In the reconstituted system the recombinant protein, exhibited high transport affinity for CoA, dephospho-CoA, ADP and adenosine 3',5'-diphosphate (PAP). The main physiological role of SLC25A42 is to import CoA into mitochondria in exchange for intra-mitochondrial adenine nucleotides and/or PAP. The export of PAP out of the mitochondria is crucial, since the catabolism of this cytotoxic molecule, produced in the organelles by the transfer of the 4'-phosphopantetheine prosthetic group of CoA to ACP, takes place in the cytosol. This is the first time that a mitochondrial carrier for CoA and PAP has been identified and characterized biochemically

The human SLC25A42 protein, ortholog of mitochondrial carrier Leu5p of S. cerevisiae, transports Coenzyme A and Adenosine 3’, 5’- diphosphate

The human SLC25A42 protein, ortholog of mitochondrial carrier Leu5p of S. cerevisiae, transports Coenzyme A and Adenosine 3’,5’-diphosphate G. Fiermonte, E. Paradies, S. Todisco, C.M.T. Marobbio, M.A Di Noia, and F. Palmieri Department of Pharmaco-Biology, Laboratory and Molecular Biology, University of Bari, Bari, Italy The essential cofactor Coenzyme A (CoA) is required in many intra-mitochondrial metabolic pathways. The CoA is synthesized outside the mitochondrial matrix, therefore must be transported into mitochondria. In S. cerevisiae, the mitochondrial carrier Leu5p is involved in the accumulation of CoA in the mitochondrial matrix. In fact, deletion of LEU5 (leu5) causes a reduction of mitochondrial coenzyme A (CoA) levels and growth defect on YP supplemented with glycerol or other non fermentative carbon sources. The closest relatives of Leu5p in human are SLC25A16 (37% identity) and SLC25A42 (31% identity). In this study we provide direct evidence that SLC25A42 is a novel transporter of CoA. SLC25A42 is localized in the mitochondrial inner membrane and is highly expressed in virtually all tissues. This protein was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles, and shown to transport CoA, dephospho-CoA, Adenosine 3’,5’-diphosphate (PAP), and (deoxy)adenine nucleotides with high specificity and by a counter-exchange mechanism. The expression of SLC25A42 protein in LEU5 cells fully restores the phenotype of the LEU5 strain, indicating that the main function of both proteins is probably to catalyze the entry of CoA into mitochondria in exchange for adenine nucleotides and PAP