The Mitochondrial Citrate Transport Protein

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The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleoprotein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. . Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

Section: Discussionmentioning

confidence: 99%

Identification of the Pore-lining Residues of the BM2 Ion Channel Protein of Influenza B Virus

Soto

Ohigashi

et al. 2008

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“…Construction, Overexpression, and Isolation of Single-Cys CTP Variants-Single-Cys CTP variants were prepared as previously described (14). Briefly, single-Cys CTP genes were constructed using the QuikChange site-directed mutagenesis kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…With this background in mind, the current studies focused on characterizing the kinetic properties of a panel of singleCys mutants that we had previously constructed based on their predicted importance in the CTP mechanism (11,14,18). We identified ten mutations that display both substantially elevated K m values and reduced V max values, nine of which localize to two topographically distinct clusters within the CTP structure.…”

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confidence: 99%

“…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 of single-Cys mutants to MTS reagents, in both the absence and presence of citrate, to identify those residues in transmembrane domains III and IV that line the substrate translocation pathway (13)(14)(15); (iii) developed a detailed homology model of the three-dimensional structure of the CTP (16) based on the crystal structure of the mitochondrial ADP/ATP carrier (17); and (iv) superimposed our functional data onto this homology model to delineate substantial portions of the translocation pathway within the structure (15,16).…”

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confidence: 99%

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

Ma¹,

Remani

Sun

et al. 2007

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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...

“…Preparation, Expression, and Isolation of Single-Cys CTP Mutants-The nine single-Cys CTP-binding site mutants were constructed using the QuikChange site-directed mutagenesis kit and cloned into storage (NovaBlue) and expression (BL21(DE3)) hosts as described previously (20,23). Expression was induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

The Yeast Mitochondrial Citrate Transport Protein

Aluvila

Kotaria

Sun

et al. 2010

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The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [ 14 C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [ 14 C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.Citrate is a prominent intermediate in both carbohydrate and lipid metabolism. Once it is formed within the mitochondrial matrix as part of the tricarboxylic acid cycle, it is then either processed by the cycle enzymes to generate NADH and FADH 2 leading ultimately to the formation of ATP, or it can be transported out of the mitochondrial matrix across the inner membrane and into the intermembrane space via the mitochondrial inner membrane citrate transport protein (CTP) 2 (1, 2). Citrate then passively diffuses through a voltage-dependent anion-selective channel within the outer membrane, into the cytoplasm, where it is broken down by citrate lyase to acetylCoA and oxaloacetate. The resulting acetyl-CoA represents a prime carbon source fueling fatty acid, triacylglycerol, and sterol biosyntheses (3-6). Consequently, the CTP is critical to the energy metabolism of eukaryotic cells.The mitochondrial CTP catalyzes an obligatory exchange of the dibasic form of tricarboxylates (i.e. citrate and isocitrate) either for each other in yeast (7,8) or for dicarboxylates or phosphoenolpyruvate in higher eukaryotes (1, 2). Its function is altered in several diseases, including cancer (9) and diabetes (10), and it is thought to figure prominently in the abnormal bioenergetics observed with these pathologies. In terms of its function in the ...