Reply from Conover

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...

Section: Measurement Of the Effect Of Mts Reagents On Reconstituted Cmentioning

confidence: 99%

mentioning

confidence: 99%

The Yeast Mitochondrial Citrate Transport Protein

Kaplan¹,

Mayor²,

Brauer³

et al. 2000

“…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)(3)(4)(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.…”

mentioning

confidence: 99%

Identification of the Substrate Binding Sites within the Yeast Mitochondrial Citrate Transport Protein

Ma¹,

Remani

Sun

et al. 2007

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...

“…malate or succinate), or phosphoenolpyruvate (1). The CTP occupies a critical position in intermediary metabolism, because the resulting cytoplasmic citrate serves as a carbon source, which fuels both the fatty acid and the sterol biosynthetic pathways, and provides a source of NAD ϩ (via the concerted actions of ATP-citrate lyase and malate dehydrogenase) for the glycolytic pathway (2)(3)(4)(5). Due to its prominent role, the CTP has been intensively studied.…”

mentioning

confidence: 99%

The Mitochondrial Citrate Transport Protein

Kotaria

Mayor

et al. 2004

The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine. A cysteine-less CTP that retains wildtype functional properties, served as the starting template. The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function. These rate constants varied by over five orders of magnitude. With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137. Based on the pattern of accessibility we conclude that residues 123-137 exist as an ␣-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the ␣-helical secondary structure may extend to residue 113. Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII dimer interface has been developed.