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 Km of 207 μM for TTP, 404 μM for UTP and 435 μM 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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Research Article|
December 23 2005
Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization
Carlo Marya Thomas Marobbio;
Carlo Marya Thomas Marobbio
*Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy
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Maria Antonietta Di Noia;
Maria Antonietta Di Noia
*Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy
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Ferdinando Palmieri
Ferdinando Palmieri
1
*Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy
†CNR Institute of Biomembranes and Bioenergetics, Via Orabona 4, 70125 Bari, Italy
1To whom correspondence should be addressed (email fpalm@farmbiol.uniba.it).
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Publisher: Portland Press Ltd
Received:
August 05 2005
Revision Received:
September 28 2005
Accepted:
September 29 2005
Accepted Manuscript online:
September 29 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (2): 441–446.
Article history
Received:
August 05 2005
Revision Received:
September 28 2005
Accepted:
September 29 2005
Accepted Manuscript online:
September 29 2005
Citation
Carlo Marya Thomas Marobbio, Maria Antonietta Di Noia, Ferdinando Palmieri; Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization. Biochem J 15 January 2006; 393 (2): 441–446. doi: https://doi.org/10.1042/BJ20051284
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