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.

To search for a genetic marker for type 2 Gaucher's disease (acute neuronopathic form), we compared the nucleotide sequence of a cloned glucocerebrosidase gene from a patient with Gaucher's disease with a normal gene. We found only a single base substitution (T----C) in exon X. This mutation results in the substitution of proline for leucine in position number 444 and produces a new cleavage site for the NciI restriction endonuclease. We analyzed NciI enzymatic digests of genomic DNA from 20 patients with type 1, 5 with type 2, and 11 with type 3 Gaucher's disease, and 29 normal controls for a restriction-fragment-length polymorphism (RFLP). Four of 5 patients with type 2 disease and all 11 with type 3 disease had at least one allele with the mutation. Two of 5 patients with type 2 disease and 7 of 11 with type 3 were homozygous for this mutation. Only 4 of 20 patients with type 1 Gaucher's disease had the mutant allele and were heterozygous for it. None of the 29 normal controls had the mutant allele. The high frequency of this mutation (444leucine----proline) in patients with neuronopathic Gaucher's disease, detectable by the NciI RFLP, may be of value in the identification of patients who will have the neurologic sequelae of Gaucher's disease.

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...

The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an ␣-helix. The citrate transport protein (i.e. CTP) 1 from mammalian mitochondria catalyzes an electroneutral exchange across the inner mitochondrial membrane of a tricarboxylate (i.e. citrate, isocitrate, cis-aconitate) plus a proton, for either another tricarboxylate-H ϩ , a dicarboxylate (i.e. malate, succinate), or phosphoenolpyruvate (1). Following the efflux of citrate from the mitochondrial matrix via the CTP, cytoplasmic citrate serves as both a carbon precursor for the fatty acid and the sterol biosynthetic pathways, as well as a source of NAD ϩ (via the concerted actions of ATP-citrate lyase and malate dehydrogenase) for the glycolytic pathway (2-5). Because of the central role of the CTP in higher eukaryotic metabolism, the transporter has been extensively studied. Thus, it has been purified (6, 7), kinetically characterized (8), cloned (9), and overexpressed (10). More recently we have extended our studies of the CTP to the yeast homologue of the higher eukaryotic protein (11). An advantage afforded by the yeast mitochondrial CTP is that following overexpression in Escherichia coli and subsequent isolation, CTP function can be reconstituted with high specific activity. Thus, the yeast CTP represents ideal material with which to conduct a comprehensive structure/function analysis. With this goal in mind, we recently constructed a Cys-less CTP that displays near native functional properties. Moreover, we have demonstrated that upon isolation, both the isolated wild-type and Cys-less CTPs exist as functional dimers.3 Based on hydropathy analysis, each monomer of the homodimer is predicted to contain six membrane-spanning domains (11).Within the CTP, transmembrane domain IV (i.e. TMDIV) is of particular interest for several reasons. First, this domain contains two arginine residues (Arg-181 and -189). We have previously demonstrated that CTP function requires the presence of positive charge at both sites.2 Second, TMDIV can be modeled as an ␣-helix with both arginines residing on the same face of the helix, thereby providing a polar domain, which might conceivably be accessible to citrate and provide a partial neutra...

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