Biochemical and Biophysical Research Communications
Cell-free synthesis, reconstitution, and characterization of a mitochondrial dicarboxylate–tricarboxylate carrier of Plasmodium falciparum
Highlights
► We isolated a cDNA encoding an MCF-like protein, PfDTC, from Plasmodium falciparum. ► PfDTC was synthesized with a cell-free system and reconstituted in proteoliposomes. ► The reconstituted PfDTC showed substrate-specific membrane transport activity. ► PfDTC is likely required for the branched-type TCA metabolism of the malaria parasite.
Introduction
Most organisms have evolved a canonical cyclic pathway of tricarboxylic acid (TCA) metabolism, although there are some exceptions [1]. For example, the malaria parasite, Plasmodium falciparum, was recently shown to operate a noncanonical branched-type TCA metabolic pathway in its mitochondria [2]. It was suggested that this protozoan does not depend on TCA metabolism for the generation of ATP, but rather relies on its unique TCA metabolism for more limited functions such as provision of a precursor, succinyl-CoA, for heme biosynthesis [2]. The starting substrate and end product of the branched TCA metabolism were proposed to be oxoglutarate and malate, respectively. It is therefore important to clarify the transporter function that facilitates efficient uptake of oxoglutarate and removal of malate across the mitochondrial inner membrane (IM).
The transport of molecules across the mitochondrial IM is highly selective, so that an effective barrier exists between the cytosol and the mitochondrial matrix. Members of the mitochondrial carrier family (MCF) of proteins, which is the largest family of transporters, facilitate the selective transport of most solutes across the IM [3]. Analysis of the genome database for P. falciparum suggested that it encodes nine MCF homologs [4], with one MCF-like protein being predicted to transport tricarboxylic or dicarboxylic acid intermediates of the TCA pathway [2].
One of the bottlenecks in biochemical analysis of P. falciparum proteins has been the lack of an efficient method for protein preparation. The parasite has one of the most A/T-rich genomes known (76.3% in exon regions) [4], with the A/T-biased codon usage having been assumed to affect the expression of encoded proteins in bacterial recombinant systems. A wheat germ cell-free (CF) system was recently shown to be a promising alternative for the production of P. falciparum proteins [5]. CF systems also provide an effective tool for the production of transmembrane proteins (TMPs) [6], [7], with a variety of modified CF synthesis methods for TMP production having been described [8], [9], [10], [11]. An apicoplast phosphate translocator from P. falciparum has been synthesized and characterized with the use of a CF system [12].
To gain insight into TCA metabolism in the mitochondria of P. falciparum, we have now isolated and characterized a predicted dicarboxylate–tricarboxylate carrier (DTC) homolog of this parasite with the use of a liposome-supplemented CF system. We clarified the substrate specificity of this protein, designated PfDTC, which has implications for the nature of the TCA pathway in the parasite.
Section snippets
Plasmid construction
Complementary DNA fragments encoding PfDTC (PlasmoDB ID: PF08_0031) and AtDTC (TAIR ID: AT5G19760) were amplified by the polymerase chain reaction (PCR) from a cDNA prepared from P. falciparum parasite rich in schizonts [5] or Arabidopsis thaliana, respectively, with the primer sets PfDTC-SpeI (5′-CCACTAGTAACAATGGACAGAGATATAGCTAAATATG-3′) and PfDTC-SalI (5′-CCGTCGACTTAAGAAATTTTTTTTAAAAGATTAT-3′) for PfDTC and AtDTC-SpeI (5′-CGACTAGTAACAATGGCGGAAGAGAAGAAAGCT-3′) and AtDTC-SalI
Isolation of DTC cDNA from P. falciparum
The P. falciparum genome has been predicted to encode nine MCF proteins [4], but the detailed sequence information has not yet been released. We therefore surveyed the P. falciparum database to identify gene sequences that encode MCF-like proteins and found 12 candidate cDNAs (Table 1). Five of the 12 predicted proteins showed significant sequence similarity to known MCF proteins, whereas the remaining seven gene products could not be assigned to any of the known MCF subfamilies. We next
Acknowledgments
We thank Y. Endo and other members of the Cell-Free Science and Technology Research Center of Ehime University for support and helpful discussion.
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