Abstract
The peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 has very recently been found to transport NAD+, NADH, AMP and ADP. In this work we have reinvestigated the substrate specificity and the transport properties of PXN by using a wide range of potential substrates. Heterologous expression in bacteria followed by purification, reconstitution in liposomes, and uptake and efflux experiments revealed that PNX transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3′, 5′-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport and was strongly inhibited by pyridoxal 5′-phosphate, bathophenanthroline and tannic acid. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Agrimi G, Di Noia MA, Marobbio CMT, Fiermonte G, Lasorsa FM, Palmieri F (2004) Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution. Biochem J 379:183–190
Agrimi G, Russo A, Scarcia P, Palmieri F (2012) The human gene SLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD and NAD+. Biochem J 443:241–247
Aluvila S, Kotaria R, Sun J, Mayor JA, Walters DE, Harrison DHT, Kaplan RS (2010) The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity. J Biol Chem 285:27314–27326
Bedhomme M, Hoffmann M, McCarthy EA, Gambonnet B, Moran RG, Rebeille F, Ravanel S (2005) Folate metabolism in plants: an Arabidopsis homolog of the mammalian mitochondrial folate transporter mediates folate import into chloroplasts. J Biol Chem 280:34823–34831
Bernhardt K, Wilkinson S, Weber APM, Linka N (2012) A peroxisomal carrier delivers NAD+ and contributes to optimal fatty acid degradation during storage oil mobilization. Plant J 69:1–13
Briggs C, Mincone L, Wohlrab H (1999) Replacements of basic and hydroxyl amino acids identify structurally and functionally sensitive regions of the mitochondrial phosphate transport protein. Biochemistry 38:5096–5102
Cappello AR, Curcio R, Miniero DV, Stipani I, Robinson AJ, Kunji ERS, Palmieri F (2006) Functional and structural role of amino acid residues in the even-numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate carrier. J Mol Biol 363:51–62
Cappello AR, Miniero DV, Curcio R, Ludovico A, Daddabbo L, Stipani I, Robinson AJ, Kunji ERS, Palmieri F (2007) Functional and structural role of amino acid residues in the odd-numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate carrier. J Mol Biol 369:400–412
Carrie C, Murcha MW, Millar AH, Smith SM, Whelan J (2007) Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana aretargeted either to peroxisomes or cytosol but not to mitochondria. Plant Mol Biol 63(1):97–108
de Lucas JR, Indiveri C, Tonazzi A, Perez P, Giangregorio N, Iacobazzi V, Palmieri F (2008) Functional characterisation of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif. Mol Membr Biol 25:152–163
Echtay KS, Bienengraeber M, Klingenberg M (2001) Role of intrahelical arginine residues in functional properties of uncoupling protein (UCP1). Biochemistry 40:5243–5248
Eubel H, Meyer EH, Taylor NL, Bussell JD, O’Toole N, Heazlewood JL, Castleden I, Small ID, Smith SM, Millar AH (2008) Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes. Plant Physiol 148:1809–1829
Fiermonte G, Walker JE, Palmieri F (1993) Abundant bacterial expression and reconstitution of an intrinsic membrane transport protein from bovine mitochondria. Biochem J 294:293–299
Fiermonte G, Dolce V, Palmieri L, Ventura M, Runswick MJ, Palmieri F, Walker JE (2001) Identification of the human mitochondrial oxodicarboxylate carrier: bacterial expression, reconstitution, functional characterization, tissue distribution, and chromosomal location. J Biol Chem 276:8225–8230
Fiermonte G, Dolce V, David L, Santorelli FM, Dionisi-Vici C, Palmieri F, Walker JE (2003) The mitochondrial ornithine transporter: bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms. J Biol Chem 278:32778–32783
Fiermonte G, Paradies E, Todisco S, Marobbio CMT, Palmieri F (2009) A novel member of solute carrier family 25 (SLC25A42) is a transporter of coenzyme a and adenosine 3′,5′-diphosphate in human mitochondria. J Biol Chem 284:18152–18159
Floyd S, Favre C, Lasorsa FM, Leahy M, Trigiante G, Stroebel P, Marx A, Loughran G, O’Callaghan K, Marobbio CMT, Slotboom DJ, Kunji ERS, Palmieri F, O’Connor R (2007) The IGF-I-mTOR signaling pathway induces the mitochondrial pyrimidine nucleotide carrier to promote cell growth. Mol Biol Cell 18:3545–3555
Fukao Y, Hayashi Y, Mano S, Hayashi M, Nishimura M (2001) Developmental analysis of a putative ATP/ADP carrier protein localized on glyoxysomal membranes during the peroxisome transition in pumpkin cotyledons. Plant Cell Physiol 42:835–841
Fulda M, Shockey J, Werber M, Wolter FP, Heinz E (2002) Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid β-oxidation. Plant J 32(1):93–103
Fulda M, Schnurr J, Abbadi A, Heinz E, Browse J (2004) Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana. Plant cell 16(2):394–405
Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N, Carde J-P, Bryce JH et al (2001) Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid β-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant J 28(1):1–12
Giangregorio N, Tonazzi A, Console L, Indiveri C, Palmieri F (2010) Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: insight into the molecular mechanism of transport. Biochim Biophys Acta 1797:839–845
Heidkämper D, Müller V, Nelson DR, Klingenberg M (1996) Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations on transport and the four ATP versus ADP exchange modes. Biochemistry 35:16144–16152
Hoyos ME, Palmieri L, Wertin T, Arrigoni R, Polacco JC, Palmieri F (2003) Identification of a mitochondrial transporter for basic amino acids in Arabidopsis thaliana by functional reconstitution into liposomes and complementation in yeast. Plant J 33:1027–1035
Indiveri C, Giangregorio N, Iacobazzi V, Palmieri F (2002) Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136. Biochemistry 41:8649–8656
Lawrence SA, Hackett JC, Moran RG (2011) Tetrahydrofolate recognition by the mitochondrial folate transporter. J Biol Chem 286:31480–31489
Lindhurst MJ, Fiermonte G, Song S, Struys E, De Leonardis F, Schwartzberg PL, Chen A, Castegna A, Verhoeven N, Mathews CK, Palmieri F, Biesecker LG (2006) Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia. Proc Nat Acad Sci U S A 103:15927–15932
Linka N, Theodoulou FL, Haslam RP, Linka M, Napier JA, Neuhaus HE, Weber APM (2008) Peroxisomal ATP import is essential for seedling development in Arabidopsis thaliana. Plant Cell 20:3241–3257
Mano S, Nakamori C, Fukao Y, Araki M, Matsuda A, Kondo M, Nishimura M (2011) A defect of peroxisomal membrane protein 38 causes enlargement of peroxisomes. Plant Cell Physiol 52:2157–2172
Marobbio CMT, Agrimi G, Lasorsa FM, Palmieri F (2003) Identification and functional reconstitution of yeast mitochondrial carrier for S-adenosylmethionine. EMBO J 22:5975–5982
Marobbio CMT, Di Noia MA, Palmieri F (2006) Identification of the mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization. Biochem J 393:441–446
Miniero DV, Cappello AR, Curcio R, Ludovico A, Daddabbo L, Stipani I, Robinson AJ, Kunji ERS, Palmieri F (2011) Functional and structural role of amino acid residues in the matrix α-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier. Biochim Biophys Acta 1807:302–310
Monné M, Miniero V, Daddabbo L, Robinson AJ, Kunji ERS, Palmieri F (2012) The substrate specificity of the two mitochondrial ornithine carriers can be swapped by a single mutation in the substrate binding site. J Biol Chem 287:7925–7934
Palmieri F (2012) The mitochondrial transporter family SLC25: identification, properties and physiopathology. Mol Aspects Med (in press)
Palmieri F, Pierri CL (2010a) Mitochondrial metabolite transport. Essays Biochem 47:37–52
Palmieri F, Pierri CL (2010b) Structure and function of mitochondrial carriers - Role of the transmembrane helix P and G residues in the gating and transport mechanism. FEBS Lett 584:1931–1939
Palmieri F, Indiveri C, Bisaccia F, Iacobazzi V (1995) Mitochondrial metabolite carrier proteins: purification, reconstitution and transport studies. Methods Enzymol 260:349–369
Palmieri L, Agrimi G, Runswick MJ, Fearnley IM, Palmieri F, Walker JE (2001a) Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate. J Biol Chem 276:1916–1922
Palmieri L, Rottensteiner H, Girzalsky W, Scarcia P, Palmieri F, Erdmann R (2001b) Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter. EMBO J 20:5049–5059
Palmieri F, Agrimi G, Blanco E, Castegna A, Di Noia MA, Iacobazzi V, Lasorsa FM, Marobbio CMT, Palmieri L, Scarcia P, Todisco S, Vozza A, Walker J (2006a) Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins. Biochim Biophys Acta 1757:1249–1262
Palmieri L, Arrigoni R, Blanco E, Carrari F, Zanor MI, Studart-Guimareas C, Fernie AR, Palmieri F (2006b) Molecular identification of an Arabidopsis thaliana S-adenosylmethionine transporter: analysis of organ distribution, bacterial expression, reconstitution into liposomes and functional characterization. Plant Physiol 142:855–865
Palmieri L, Picault N, Arrigoni R, Besin E, Palmieri F, Hodges M (2008) Molecular identification of three Arabidopsis thaliana mitochondrial dicarboxylate carrier isoforms: organ distribution, bacterial expression, reconstitution into liposomes and functional characterization. Biochem J 410:621–629
Palmieri F, Rieder B, Ventrella A, Blanco E, Do PT, Nunes-Nesi A, Trauth AU, Fiermonte G, Tjaden J, Agrimi G et al (2009) Molecular identification and functional characterisation of Arabidopsis thaliana mitochondrial and chloroplastic NAD+ carrier proteins. J Biol Chem 284:31249–31259
Palmieri F, Pierri CL, De Grassi A, Nunes-Nesi A, Fernie AR (2011) Evolution, structure and function of mitochondrial carriers: a review with new insights. Plant J 66:161–181
Pracharoenwattana I, Cornah JE, Smith SM (2005) Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell Online 17(7):2037, Am Soc Plant Biol
Reumann S, Quan S, Aung K, Yang P, Manandhar-Shrestha K, Holbrook D, Linka N, Switzenberg R, Wilkerson CG, Weber AP, Olsen LJ, Hu J (2009) In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes. Plant Physiol 150:125–143
Robinson A, Overy C, Kunji E (2008) The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proc Natl Acad Sci U S A 105:17766–17771
Stipani V, Cappello AR, Daddabbo L, Natuzzi D, Miniero DV, Stipani I, Palmieri F (2001) The mitochondrial oxoglutarate carrier: cysteine-scanning mutagenesis of transmembrane domain IV and sensitivity of cys mutants to sulphydryl reagents. Biochemistry 40:15805–15810
Titus SA, Moran RG (2000) Retrovirally mediated complementation of the glyB phenotype: cloning of a human gene encoding the carrier for entry of folates into mitochondria. J Biol Chem 275:36811–36817
Todisco S, Agrimi G, Castegna A, Palmieri F (2006) Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae. J Biol Chem 281:1524–1531
Tonazzi A, Giangregorio N, Indiveri C, Palmieri F (2005) Identification by site-directed mutagenesis and chemical modification of three vicinal cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter. J Biol Chem 280:19607–19612
Tonazzi A, Console L, Giangregorio N, Indiveri C, Palmieri F (2012) Identification by site-directed mutagenesis of a hydrophobic binding site of the mitochondrial carnitine/acylcarnitine carrier involved in the interaction with acyl groups. Biochim Biophys Acta 1817:697–704
Tzagoloff A, Jang J, Glerum DM, Wu M (1996) FLX1 codes for a carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria. J Biol Chem 271:7392–7397
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Agrimi, G., Russo, A., Pierri, C.L. et al. The peroxisomal NAD+ carrier of Arabidopsis thaliana transports coenzyme A and its derivatives. J Bioenerg Biomembr 44, 333–340 (2012). https://doi.org/10.1007/s10863-012-9445-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10863-012-9445-0