Mitochondrial Citrate Transport Protein Research Articles

α-Ketoglutarate supplementation and NAD+ modulation enhance metabolic rewiring and radiosensitization in SLC25A1 inhibited cancer cells

Metabolic rewiring is the result of the increasing demands and proliferation of cancer cells, leading to changes in the biological activities and responses to treatment of cancer cells. The mitochondrial citrate transport protein SLC25A1 is involved in metabolic reprogramming offering a strategy to induce metabolic bottlenecks relevant to radiosensitization through the accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) upon SLC25A1 inhibition (SLC25A1i). Previous studies have revealed the comparative effects of SLC25A1i or cell-permeable D-2HG (octyl-D-2HG) treatments on DNA damage induction and repair, as well as on energy metabolism and cellular function, which are crucial for the long-term survival of irradiated cells. Here, α-ketoglutarate (αKG), the precursor of D-2HG, potentiated the effects observed upon SLC25A1i on DNA damage repair, cell function and long-term survival in vitro and in vivo, rendering NCI-H460 cancer cells more vulnerable to ionizing radiation. However, αKG treatment alone had little effect on these phenotypes. In addition, supplementation with nicotinamide (NAM), a precursor of NAD (including NAD+ and NADH), counteracted the effects of SLC25A1i or the combination of SLC25A1i with αKG, highlighting a potential importance of the NAD+/NADH balance on cellular activities relevant to the survival of irradiated cancer cells upon SLC25A1i. Furthermore, inhibition of histone lysine demethylases (KDMs), as a major factor affected upon SLC25A1i, by JIB04 treatment alone or in combination with αKG supplementation phenocopied the broad effects on mitochondrial and cellular function induced by SLC25A1i. Taken together, αKG supplementation potentiated the effects on cellular processes observed upon SLC25A1i and increased the cellular demand for NAD to rebalance the cellular state and ensure survival after irradiation. Future studies will elucidate the underlying metabolic reprogramming induced by SLC25A1i and provide novel therapeutic strategies for cancer treatment.

FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress

Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

Accumulation of oncometabolite D-2-Hydroxyglutarate by SLC25A1 inhibition: A metabolic strategy for induction of HR-ness and radiosensitivity

Oncogenic mutations in metabolic genes and associated oncometabolite accumulation support cancer progression but can also restrict cellular functions needed to cope with DNA damage. For example, gain-of-function mutations in isocitrate dehydrogenase (IDH) and the resulting accumulation of the oncometabolite D-2-hydroxyglutarate (D-2-HG) enhanced the sensitivity of cancer cells to inhibition of poly(ADP-ribose)-polymerase (PARP)1 and radiotherapy (RT). In our hand, inhibition of the mitochondrial citrate transport protein (SLC25A1) enhanced radiosensitivity of cancer cells and this was associated with increased levels of D-2-HG and a delayed repair of radiation-induced DNA damage. Here we aimed to explore the suggested contribution of D-2-HG-accumulation to disturbance of DNA repair, presumably homologous recombination (HR) repair, and enhanced radiosensitivity of cancer cells with impaired SLC25A1 function. Genetic and pharmacologic inhibition of SLC25A1 (SLC25A1i) increased D-2-HG-levels and sensitized lung cancer and glioblastoma cells to the cytotoxic action of ionizing radiation (IR). SLC25A1i-mediated radiosensitization was abrogated in MEFs with a HR-defect. D-2-HG-accumulation was associated with increased DNA damage and delayed resolution of IR-induced γH2AX and Rad51 foci. Combining SLC25A1i with PARP- or the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs)-inhibitors further potentiated IR-induced DNA damage, delayed DNA repair kinetics resulting in radiosensitization of cancer cells. Importantly, proof of concept experiments revealed that combining SLC25A1i with IR without and with PARPi also reduced tumor growth in the chorioallantoic membrane (CAM) model in vivo. Thereby SLC25A1i offers an innovative strategy for metabolic induction of context-dependent lethality approaches in combination with RT and clinically relevant inhibitors of complementary DNA repair pathways.

Quantitative metabolic flux analysis reveals an unconventional pathway of fatty acid synthesis in cancer cells deficient for the mitochondrial citrate transport protein

The mitochondrial citrate transport protein (CTP), encoded by SLC25A1, accommodates bidirectional trafficking of citrate between the mitochondria and cytosol, supporting lipid biosynthesis and redox homeostasis. Genetic CTP deficiency causes a fatal neurodevelopmental syndrome associated with the accumulation of L- and D-2-hydroxyglutaric acid, and elevated CTP expression is associated with poor prognosis in several types of cancer, emphasizing the importance of this transporter in multiple human pathologies. Here we describe the metabolic consequences of CTP deficiency in cancer cells. As expected from the phenotype of CTP-deficient humans, somatic CTP loss in cancer cells induces broad dysregulation of mitochondrial metabolism, resulting in accumulation of lactate and of the L- and D- enantiomers of 2-hydroxyglutarate (2HG) and depletion of TCA cycle intermediates. It also eliminates mitochondrial import of citrate from the cytosol. To quantify the impact of CTP deficiency on metabolic flux, cells were cultured with a set of 13C-glucose and 13C-glutamine tracers with resulting data integrated by metabolic flux analysis (MFA). CTP-deficient cells displayed a major restructuring of central carbon metabolism, including suppression of pyruvate dehydrogenase (PDH) and induction of glucose-dependent anaplerosis through pyruvate carboxylase (PC). We also observed an unusual lipogenic pathway in which carbon from glucose supplies mitochondrial production of alpha-ketoglutarate (AKG), which is then trafficked to the cytosol and used to supply reductive carboxylation by isocitrate dehydrogenase 1 (IDH1). The resulting citrate is cleaved to produce lipogenic acetyl-CoA, thereby completing a novel pathway of glucose-dependent reductive carboxylation. In CTP deficient cells, IDH1 inhibition suppresses lipogenesis from either glucose or glutamine, implicating IDH1 as a required component of fatty acid synthesis in states of CTP deficiency.

Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

The Yeast Mitochondrial Citrate Transport Protein: MOLECULAR DETERMINANTS OF ITS SUBSTRATE SPECIFICITY

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.

Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach.

The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter's two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5'-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.

Transport Inhibitors Cause Conformational Changes in the Yeast Mitochondrial Citrate Transport Protein Reconstituted in Liposomes as Demonstrated by EPR Spectroscopy

In order to directly observe conformational change in the mitochondrial citrate transport protein (CTP), we measured, in the presence and absence of inhibitors, the EPR spectra of spin-labeled single-Cys CTP mutants that were reconstituted in liposomes. We selected spin-label locations to report on substrate binding sites 1 and 2 (i.e., 187, 183, and 179), binding site 2 (39), TMDIII pointing away from the transport pathway (118), and a matrix-facing hydrophilic loop (47). In the absence of inhibitor, the EPR lineshapes show residue-dependent variations in mobility. Addition of external 1,2,3-benzenetricarboxylate (BTC), the defining inhibitor of the CTP, caused a modest, residue-dependent decrease in the mobile component and a concomitant increase in the immobile component. Addition of compound 792949, a novel, purely competitive inhibitor that we previously identified via high throughput in silico screening using the homology-modeled CTP in its cytosolic-facing conformation, yielded EPR spectra that contain a substantial increase in the immobile component at each location. We conclude that the two inhibitors cause CTP to assume different conformations, which vary significantly in their extent of immobilization.Surprisingly, in contrast to the large immobilizing effect observed upon the addition of extraliposomal inhibitor 792949, the inclusion of intraliposomal inhibitor caused only a minor spectral change. This observation indicates that binding of 792949 to CTP from the internal surface of the proteoliposomes (i.e., the matrix-facing conformation) occurs to a much lesser extent than does binding to CTP from the external surface (i.e., the cytosolic-facing conformation). We conclude that external 792949 affects spin-label mobility at both monomers within the functional homodimer suggesting a tight coordination of the two monomers. Supported by NIH grant GM-054642 to R.S.K.

Inhibitors of the Mitochondrial Citrate Transport Protein: Validation of the Role of Substrate Binding Residues and Discovery of the First Purely Competitive Inhibitor

The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (K(ic)) of 0.12 +/- 0.02 mM and an uncompetitive inhibition constant (K(iu)) of 3.04 +/- 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the K(ic) values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62- to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the K(m) for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, K(m) approximates the K(d) (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport.

The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate.

The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH(2)CH(2)NH(3) (+)) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.

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

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 > or = 98% and resultant catalytic efficiencies that were reduced by > or = 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.

Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier

Conjugated linoleic acid (CLA) is able to reduce adiposity by affecting lipid metabolism. In particular, CLA administration to mice reduces body fat mass with a concomitant lipid accumulation in the liver. We investigated the effects of CLA on the activity of the mitochondrial citrate carrier (CIC), which is implicated in hepatic lipogenesis. The transport activity of the CIC, measured both in intact mitochondria and in the proteoliposomes, progressively increased with the duration of CLA feeding. An increase in the CIC activity of approximately 1.7-fold was found in 16 week CLA-treated mice with respect to control animals. A kinetic analysis showed a 1.6-fold increase in the V(max) of citrate transport but no change in the K(m) value. Western blot experiments revealed an increase of approximately 1.7-fold in the expression of CIC after CLA treatment. A strict correlation between the increase in CIC activity and the stimulation of the cytosolic lipogenic enzymes was also found. These data indicate that the CIC may play a role in the onset of hepatic steatosis in CLA-fed mice by supplying the carbon source for de novo fatty acid synthesis.

The Yeast Mitochondrial Citrate Transport Protein: CHARACTERIZATION OF TRANSMEMBRANE DOMAIN III RESIDUE INVOLVEMENT IN SUBSTRATE TRANSLOCATION

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to hydrophilic, cysteine-specific methanethiosulfonate reagents, enabled identification of the water-accessible surface of this domain and suggested its potential participation in the formation of a portion of the substrate translocation pathway. To evaluate this idea, we conducted a detailed characterization of the functional properties of 20 TMDIII single-Cys substitution mutants. Kinetic studies indicate that the A118C, S123C, and K134C mutants displayed a 3- to 7-fold increase in K(m). Moreover, the A118C mutation caused a doubling of the V(max) value, whereas the S123C, E131C, and K134C mutations caused V(max) to dramatically decrease, resulting in a reduction of the catalytic efficiencies of these three mutants by >97%. Examination of the ability of citrate to protect against the inhibition mediated by sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) indicated that citrate conferred significant protection of cysteines substituted at eight water-accessible locations (i.e. Gly-115, Leu-116, Gly-117, Leu-121, Ser-123, Val-127, Glu-131, and Thr-135), but not at other sites. Importantly, similar levels of protection were observed at both 4 degrees C and 20 degrees C. The temperature independence of the protection indicates that substrate binding and/or occupancy of the transport pathway sterically blocks the access of MTSES to these sites, thereby providing direct protection, without involvement of a major protein conformational change. The significance of these extensive functional investigations is discussed in terms of the three-dimensional CTP homology model that we previously developed and a new model of the dimer interface.

Analysis of the Secondary Structure of the Cys-Less Yeast Mitochondrial Citrate Transport Protein and Four Single-Cys Variants by Circular Dichroism

Utilizing cysteine scanning mutagenesis, with functional Cys-less citrate transport protein (CTP) serving as the starting template, we previously demonstrated that four single-Cys mutants located in transmembrane domains III and IV, rendered the CTP nonfunctional. The present investigations assess and quantify the secondary structure of the Cys-less CTP and the four single-Cys mutants, both in the absence and presence of citrate, via circular dichroism (CD) spectroscopy. In detergent micelles, highly purified Cys-less CTP contained approximately 50% alpha-helix and approximately 20% beta-sheet. The CD spectra of the G119C, E122C, R181C, and R189C mutants in detergent micelles were virtually superimposable with that of the functional Cys-less CTP, thereby suggesting that the wild-type residues, rather than affecting structure, may assume important mechanistic roles. Exogenously added citrate caused a significant change in the CD spectra of all solubilized CTP samples. Analyses of the spectra of the Cys-less CTP indicated an approximately 10% increase in its alpha-helical content in the presence of citrate. The conformational changes effected by the addition of substrate were less pronounced with the single-Cys mutants. Studies of the Cys-less CTP reconstituted in liposomes indicated that while the CD spectra was red-shifted, the net secondary structure of the reconstituted carrier is approximately equivalent to that of the transporter in detergent micelles, and displayed a response to added citrate. In combination, the above studies indicate that purified Cys-less CTP in either sarkosyl micelles or in liposomes, and the four inactive single-Cys mutants in sarkosyl micelles, retain native-like structure, and thus represent ideal material for detailed structural characterization.

Homology-Modeled Structure of the Yeast Mitochondrial Citrate Transport Protein

We have used homology modeling to construct a three-dimensional model of the yeast mitochondrial citrate transport protein (CTP), based on the recently published x-ray crystal structure of another mitochondrial transport protein, the ADP/ATP carrier. Superposition of the backbone traces of the homology-modeled CTP onto the crystallographically determined ADP carrier structure indicates that the CTP transmembrane domains are well modeled (i.e., root mean square deviation of 0.94 Å), whereas the loops facing the intermembrane space and the mitochondrial matrix are less certain (i.e., root mean square deviation values of 0.72–2.06 Å). The homology-modeled CTP is consistent with our earlier de novo models of the transporter's transmembrane domains, with respect to residues which face into the transport path. Importantly, the resulting model is consistent with our previous experimental data obtained from measuring reactivity of 34 single cysteine mutants in transmembrane domains 3 and 4 with methanethiosulfonate reagents. The model also points to a likely dimer interface region. In conclusion, our data help to define the substrate translocation pathway in both the modeled CTP structure and the crystallographic ADP carrier structure.

The Mitochondrial Citrate Transport Protein: PROBING THE SECONDARY STRUCTURE OF TRANSMEMBRANE DOMAIN III, IDENTIFICATION OF RESIDUES THAT LIKELY COMPRISE A PORTION OF THE CITRATE TRANSPORT PATHWAY, AND DEVELOPMENT OF A MODEL FOR THE PUTATIVE TMDIII-TMDIII′ INTERFACE

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 wild-type 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 alpha-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the alpha-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.

Models of the Transmembrane Domains of the Yeast Mitochondrial CitrateTransport Protein

We have used cysteine scanning, hydropathy analysis and molecular modeling to construct four possible models of the transmembrane helical domains of the yeast mitochondrial citrate transport protein. Models 1 and 2 invoke the formation of a translocation pathway by the six membrane-spanning alpha-helical domains that comprise each citrate transport protein monomer. Thus the homodimeric CTP (the functional form of the CTP) would contain two separate translocation pathways. Models 3 and 4 explore a novel way in which dimerization might take place, in which transmembrane domain 3 would form part of the dimer interface. This would lead to the formation of two seven-helix translocation pathways within the transporter dimer. Importantly, these studies have led to the construction of the first detailed structural models for any of the mitochondrial anion transport proteins, a family of proteins which is essential to cellular bioenergetics. Furthermore, these models suggest numerous experiments which can be carried out to further elucidate the structure of the translocation pathway through the membrane.

The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

The Yeast Mitochondrial Citrate Transport Protein: PROBING THE SECONDARY STRUCTURE OF TRANSMEMBRANE DOMAIN IV AND IDENTIFICATION OF RESIDUES THAT LIKELY COMPRISE A PORTION OF THE CITRATE TRANSLOCATION PATHWAY

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 alpha-helix. Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192. 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.

The Yeast Mitochondrial Citrate Transport Protein: PROBING THE ROLES OF CYSTEINES, Arg181, AND Arg189 IN TRANSPORTER FUNCTION

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.