The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e. Km = 0.36 mM and Vmax = 2.5 mumol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.

Integral membrane proteins of the divalent anion/Na+ symporter (DASS) family translocate dicarboxylate, tricarboxylate or sulphate across cell membranes, typically by utilizing the preexisting Na+ gradient. The molecular determinants for substrate recognition by DASS remain obscure, largely owing to the absence of any substrate-bound DASS structure. Here we present 2.8-Å resolution X-ray structures of VcINDY, a DASS from Vibrio cholerae that catalyses the co-transport of Na+ and succinate. These structures portray the Na+-bound VcINDY in complexes with succinate and citrate, elucidating the binding sites for substrate and two Na+ ions. Furthermore, we report the structures of a humanized variant of VcINDY in complexes with succinate and citrate, which predict how a human citrate-transporting DASS may interact with its bound substrate. Our findings provide insights into metabolite transport by DASS, establishing a molecular basis for future studies on the regulation of this transport process.

No abstract

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of K m values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated K m values (i.e. 5 to >300-fold). Each of these mutants displayed V max values that were reduced by >98% and resultant catalytic efficiencies that were reduced by >99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.The mitochondrial citrate transport protein (CTP) 3 is located within the inner mitochondrial membrane and catalyzes an obligatory exchange of the dibasic form of tricarboxylic acids (e.g. citrate and isocitrate) for other tricarboxylic acids or in higher eukaryotes for dicarboxylic acids (e.g. malate and succinate) or phosphoenolpyruvate (1). Once in the cytoplasm, the transported citrate serves as the prime carbon source fueling fatty acid, triacylglycerol, and cholesterol biosyntheses (2-5). In addition, the concerted action of citrate lyase and malate dehydrogenase enables the generation of NAD ϩ , a cofactor that is essential for the glycolytic pathway. Based on these roles, the CTP is considered essential for eukaryotic cell metabolism.Because of the prominent role of the CTP in cellular bioenergetics, our laboratory has conducted extensive investigations with the aim of elucidating its structure-based mechanism. Thus we have cloned (6), overexpressed (7,8), and purified (9, 10) the functional form of this transporter. Recently, employing a Cys-less yeast mitochondrial CTP construct that displays native functional properties (11) as the template, we have: (i) demonstrated that the transporter exists as a homodimer in detergent micelles (12); (ii) utilized cysteine-scanning mutagenesis combined with probing the accessibility o...