Identification of a Mitochondrial Transporter for Pyrimidine Nucleotides in Saccharomyces Cerevisiae: Bacterial Expression, Reconstitution and Functional Characterization

Overview

Journal Biochem J

Specialty Biochemistry

Date 2005 Oct 1

PMID 16194150

Citations 36

Authors

Carlo Marya Thomas Marobbio

Maria Antonietta Di Noia

Ferdinando Palmieri

Affiliations

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Abstract

Pyrimidine (deoxy)nucleoside triphosphates are required in mitochondria for the synthesis of DNA and the various types of RNA present in these organelles. In Saccharomyces cerevisiae, these nucleotides are synthesized outside the mitochondrial matrix and must therefore be transported across the permeability barrier of the mitochondrial inner membrane. However, no protein has ever been found to be associated with this transport activity. In the present study, Rim2p has been identified as a yeast mitochondrial pyrimidine nucleotide transporter. Rim2p (replication in mitochondria 2p) is a member of the mitochondrial carrier protein family having some special features. The RIM2 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes and its transport properties and kinetic parameters were characterized. It transported the pyrimidine (deoxy)nucleoside tri- and di-phosphates and, to a lesser extent, pyrimidine (deoxy)nucleoside monophosphates, by a counter-exchange mechanism. Transport was saturable, with an apparent K(m) of 207 microM for TTP, 404 microM for UTP and 435 microM for CTP. Rim2p was strongly inhibited by mercurials, bathophenanthroline, tannic acid and Bromocresol Purple, and partially inhibited by bongkrekic acid. Furthermore, the Rim2p-mediated heteroexchanges, TTP/TMP and TTP/TDP, are electroneutral and probably H+-compensated. The main physiological role of Rim2p is proposed to be to transport (deoxy)pyrimidine nucleoside triphosphates into mitochondria in exchange for intramitochondrially generated (deoxy)pyrimidine nucleoside monophosphates.

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References

Fiermonte G, Walker J, Palmieri F . Abundant bacterial expression and reconstitution of an intrinsic membrane-transport protein from bovine mitochondria. Biochem J. 1993; 294 ( Pt 1):293-9. PMC: 1134597. DOI: 10.1042/bj2940293. View

WOHLRAB H, Briggs C . Yeast mitochondrial phosphate transport protein expressed in Escherichia coli. Site-directed mutations at threonine-43 and at a similar location in the second tandem repeat (isoleucine-141). Biochemistry. 1994; 33(32):9371-5. DOI: 10.1021/bi00198a001. View

Kaplan R, Mayor J, Gremse D, Wood D . High level expression and characterization of the mitochondrial citrate transport protein from the yeast Saccharomyces cerevisiae. J Biol Chem. 1995; 270(8):4108-14. DOI: 10.1074/jbc.270.8.4108. View

Van Dyck E, Jank B, Ragnini A, Schweyen R, Duyckaerts C, Sluse F . Overexpression of a novel member of the mitochondrial carrier family rescues defects in both DNA and RNA metabolism in yeast mitochondria. Mol Gen Genet. 1995; 246(4):426-36. DOI: 10.1007/BF00290446. View

Palmieri F, Indiveri C, Bisaccia F, Iacobazzi V . Mitochondrial metabolite carrier proteins: purification, reconstitution, and transport studies. Methods Enzymol. 1995; 260:349-69. DOI: 10.1016/0076-6879(95)60150-3. View

Tzagoloff A, Jang J, Glerum D, Wu M . FLX1 codes for a carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria. J Biol Chem. 1996; 271(13):7392-7. DOI: 10.1074/jbc.271.13.7392. View

Palmieri L, Palmieri F, Runswick M, Walker J . Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein. FEBS Lett. 1996; 399(3):299-302. DOI: 10.1016/s0014-5793(96)01350-6. View

El Moualij B, Duyckaerts C, Lamotte-Brasseur J, Sluse F . Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae. Yeast. 1997; 13(6):573-81. DOI: 10.1002/(SICI)1097-0061(199705)13:6<573::AID-YEA107>3.0.CO;2-I. View

Nelson D, Felix C, Swanson J . Highly conserved charge-pair networks in the mitochondrial carrier family. J Mol Biol. 1998; 277(2):285-308. DOI: 10.1006/jmbi.1997.1594. View

10.

Fiore C, Trezeguet V, Le Saux A, Roux P, Schwimmer C, Dianoux A . The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects. Biochimie. 1998; 80(2):137-50. DOI: 10.1016/s0300-9084(98)80020-5. View

11.

Fiermonte G, Dolce V, Palmieri F . Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria. J Biol Chem. 1998; 273(35):22782-7. DOI: 10.1074/jbc.273.35.22782. View

12.

Palmieri L, Lasorsa F, Vozza A, Agrimi G, Fiermonte G, Runswick M . Identification and functions of new transporters in yeast mitochondria. Biochim Biophys Acta. 2000; 1459(2-3):363-9. DOI: 10.1016/s0005-2728(00)00173-0. View

13.

Prohl C, Pelzer W, Diekert K, Kmita H, Bedekovics T, Kispal G . The yeast mitochondrial carrier Leu5p and its human homologue Graves' disease protein are required for accumulation of coenzyme A in the matrix. Mol Cell Biol. 2001; 21(4):1089-97. PMC: 99563. DOI: 10.1128/MCB.21.4.1089-1097.2001. View

14.

Palmieri L, Rottensteiner H, Girzalsky W, Scarcia P, Palmieri F, Erdmann R . Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter. EMBO J. 2001; 20(18):5049-59. PMC: 125274. DOI: 10.1093/emboj/20.18.5049. View

15.

Palmieri L, Pardo B, Lasorsa F, Del Arco A, Kobayashi K, Iijima M . Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria. EMBO J. 2001; 20(18):5060-9. PMC: 125626. DOI: 10.1093/emboj/20.18.5060. View

16.

Palmieri L, Runswick M, Fiermonte G, Walker J, Palmieri F . Yeast mitochondrial carriers: bacterial expression, biochemical identification and metabolic significance. J Bioenerg Biomembr. 2002; 32(1):67-77. DOI: 10.1023/a:1005564429242. View

17.

Kumar A, Agarwal S, Heyman J, Matson S, Heidtman M, Piccirillo S . Subcellular localization of the yeast proteome. Genes Dev. 2002; 16(6):707-19. PMC: 155358. DOI: 10.1101/gad.970902. View

18.

Giaever G, Chu A, Ni L, Connelly C, Riles L, Veronneau S . Functional profiling of the Saccharomyces cerevisiae genome. Nature. 2002; 418(6896):387-91. DOI: 10.1038/nature00935. View

19.

Marobbio C, Vozza A, Harding M, Bisaccia F, Palmieri F, Walker J . Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate. EMBO J. 2002; 21(21):5653-61. PMC: 131080. DOI: 10.1093/emboj/cdf583. View

20.

Amutha B, Pain D . Nucleoside diphosphate kinase of Saccharomyces cerevisiae, Ynk1p: localization to the mitochondrial intermembrane space. Biochem J. 2002; 370(Pt 3):805-15. PMC: 1223228. DOI: 10.1042/BJ20021415. View