Identification of a mitochondrial transporter for pyrimidine nucleotides in Saccharomyces cerevisiae: bacterial expression, reconstitution and functional characterization

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.

Figure 1. Expression of Rim2p in E. coli and its purification

Proteins were separated by SDS-PAGE and stained with Coomassie Blue dye. Markers (‘M’; BSA, ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, carbonic anhydrase, trypsinogen, trypsin inhibitor and α-lactalbumin) are shown on the left and on the right. Lanes 1–4, E. coli CO214(DE3) containing the expression vector with (lanes 2 and 4) and without (lanes 1 and 3) the coding sequence of Rim2p. Samples were taken at the time of induction (lanes 1 and 2) and 5 h later (lanes 3 and 4). The same number of bacteria was analysed in each sample. Lane 5, purified Rim2p (6 μg) originating from bacteria shown in lane 4.

Figure 2. Homoexchange activities of various substrates in proteoliposomes reconstituted with Rim2p

Transport was initiated by adding radioactively labelled substrate (final concn. 0.2 mM) to proteoliposomes preloaded internally with the same substrate (concn. 10 mM). The reaction time was 30 min. Results are means±S.D. for at least three independent experiments.

Figure 3. Dependence of Rim2p activity on internal substrate

Proteoliposomes were preloaded internally with various substrates (concn. 10 mM). Transport was initiated by adding 0.25 mM [³H]TTP to proteoliposomes and the reaction was terminated after 45 s. Results are means±S.D. for at least three independent experiments.

Figure 4. Kinetics of [³H]TTP transport in proteoliposomes reconstituted with Rim2p

(A) Uptake of TTP. [³H]TTP (1 mM) was added to proteoliposomes containing 10 mM TTP (exchange, ■) or 10 mM NaCl and no substrate (uniport, □). (B) Efflux of [³H]TTP from proteoliposomes reconstituted in the presence of 1 mM TTP. The internal substrate pool was labelled with [³H]TTP by carrier-mediated exchange equilibration. The proteoliposomes were then passed through Sephadex G-75. The efflux of [³H]TTP was initiated by adding buffer A alone (●), buffer A and 0.5 mM p-hydroxymercuribenzoate (○), 5 mM TTP in buffer A (■) or 5 mM TTP and 0.5 mM p-hydroxymercuribenzoate in buffer A (□). Similar results were obtained in three independent experiments for both uptake and efflux of TTP.