Characterization of mitochondrial carrier proteins of malaria parasite Plasmodium falciparum based on in vitro translation and reconstitution

Abstract

Members of the mitochondrial carrier (MC) family of membrane transporters play important roles in cellular metabolism. We previously established an in vitro reconstitution system for membrane transporters based on wheat germ cell-free translation system. We have now applied this reconstitution system to the comparative analysis of MC proteins from the malaria parasite Plasmodium falciparum and Saccharomyces cerevisiae. We synthesized twelve putative P. falciparum MCs and determined the transport activities of four of these proteins including PF3D7_1037300 protein (ADP/ATP translocator), PF3D7_1004800 protein (ADP/ATP translocator), PF3D7_1202200 protein (phosphate carrier), and PF3D7_1241600 protein (S-adenosylmethionine transporter). In addition, we tested the effect of cardiolipin on the activity of MC proteins. The transport activities of the yeast MCs, ScAac2p, ScGgc1p, ScDic1p, ScPic1p, and ScSam5p, which localize to the mitochondrial inner membrane, were increased by cardiolipin supplementation, whereas that of ScAnt1p, which localizes to the peroxisome membrane, was not significantly affected. Together, this indicates that the functional properties of the reconstituted MCs reflect the lipid content of their native membranes. Except for PF3D7_1241600 protein, these P. falciparum proteins manifested cardiolipin-dependent transport activities. Immunofluorescence analysis showed that PF3D7_1241600 protein is not mainly localized to the mitochondria of P. falciparum cells. We thus revealed the functions of four MC proteins of the malaria parasite and the effects of cardiolipin on their activities.

Introduction

Malaria is a disease caused by the protozoan parasite of the genus Plasmodium and is widespread in tropical and subtropical regions. Among the five Plasmodium species that infect humans, Plasmodium falciparum causes the highest mortality, leading to half a million deaths annually [1]. Plasmodium species have distinct metabolic pathways that are essential to their growth and survival, and two organelles, the mitochondrion and the apicoplast, are critical hubs of their unique metabolisms. A number of researches have extensively focused on these organelles as they are potential targets for developing antimalarial drugs. One currently prescribed antimalarial is atovaquone, a compound that specifically affects the functions of the parasite mitochondrial respiratory chain [2,3]. Thus, it is crucial to understand the detailed function and properties of these organelles. Our group has been focusing on the mitochondrial membrane transporters of P. falciparum [4].

Many metabolic processes including the citric acid cycle, β-oxidation of fatty acids, and the urea cycle take place in the mitochondria [5]. The efficient progression of these metabolic reactions requires a rapid and highly selective exchange of metabolites between the cytosol and the mitochondrial matrix. The translocation of numerous metabolites across the inner membrane of the mitochondria is mediated by members of the mitochondrial carrier (MC) family proteins. Metabolites transported by the MC proteins are highly variable in size and structure, ranging from protons to NAD⁺ [6]. MC proteins harbor three tandem repeats of a conserved MC domain of ~100 amino acid residues that contain two transmembrane α-helices [6]. These helices possess well-conserved signature motifs, with the first helix containing PX(D/E)XX(K/R) (where X is any amino acid) on the matrix side [7] and the second containing (F/Y)(D/E)XX(K/R) on the intermembrane space side [8]. Many MC or MC-related proteins have been identified in genome databases for various eukaryotic species on the basis of these conserved sequence features. MC proteins constitute one of the largest families of solute carriers, with 58, 53, and 35 genes encoding such proteins having been identified in the genomes of Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens, respectively [6,9]. Although mitochondria are essentially ubiquitous in eukaryotic cells, the metabolic processes that take place in these organelles differ among cell types and species. The functions of MC proteins are intrinsic to such variations in mitochondrial metabolism.

For functional analysis of transport activity of MC proteins, the proteoliposome reconstitution system using recombinant MC proteins expressed in Escherichia coli has been extensively employed [10,11]. By using this method, most of S. cerevisiae MC proteins and many of A. thaliana and H. sapiens MC proteins have been identified and thoroughly characterized [6,9,12]. Although this method is well established, alternative methods are needed for the analysis of MC proteins which are difficult to express in the E. coli system. Cell-free protein synthesis systems have recently emerged as promising alternatives for membrane protein production and analysis [[13], [14], [15]]. By supplementation of a cell-free system with liposomes, we have established a functional membrane protein production system [[14], [15], [16], [17], [18]]. With the use of this approach, we previously analyzed the substrate specificity of an MC protein (PfDTC) of the malaria parasite Plasmodium falciparum [4].

Specific lipids have been found to bind to membrane proteins and thereby modulate their structure and activity. The aquaporin protein AqpZ and the ammonia channel AmtB of E. coli were recently shown to achieve structural stability through the binding of cardiolipin and phosphatidylglycerol, respectively [19]. On the other hand, human Na⁺, K⁺-ATPase was found to be stabilized, stimulated, or inhibited by the binding of phosphatidylserine, polyunsaturated phosphatidylethanolamine, and sphingomyelin, respectively [20]. Among MC proteins, the transport activities of phosphate, ADP/ATP, and carnitine carriers were shown to be stimulated by the addition of cardiolipin to the reconstitution assay system [11,21,22]. In addition, the binding of cardiolipin to ADP/ATP carriers has been confirmed by x-ray crystallography [[23], [24], [25]]. However, the effect of cardiolipin on the transport activity of other MC proteins has remained unknown.

In this study, we tested the general applicability of our transporter expression and reconstitution system for the analysis of MC protein transport activity using well-characterized MC proteins from S. cerevisiae. Then, we characterized the transport activity of four MC proteins from the malaria parasite P. falciparum and examined the effect of cardiolipin on the transport of determined substrates.