The mitochondrial tricarboxylate carrier

The tricarboxylate carrier has recently been purified from rat liver mitochondria by three distinct scientific groups using different methods. A 37–38-kDa protein has been prepared by silca gel 60 chromatography by our group (Claeys and Azzi, 1989; Glerumet al., 1990). The specific citrate transport activity of this preparation is not significantly different from that measured in mitochondria and it is inhibitable by 1,2,3-benzenetricarboxylic acid. Bisacciaet al. (1990) have reported the isolation of a 30-kDa protein by Celite 535 chromatography, and Kaplan's group (Kaplanet al., 1990) have isolated a 32.5-kDa protein by Matrex Orange, Matrex Blue, and Affi-Gel chromatography. Peptide mapping has failed to support any structural homologies between the 37–38-kDa and the 30–32.5-kD proteins. The 38-kD protein is N-terminally blocked. The peptides obtained by several cleavage procedures have been partially sequenced. Their sequence information has been used to obtain different cDNA clones by a dual approach, the polymerase chain reaction and screening of a ZAP cDNA library. The largest cDNA which could be isolated is 2,986 bp in length and contains a 1071-bp-long open reading frame and an unusually long 3 untranslated region, both of which have been completely sequenced. The protein sequence of the carrier from the first in-frame methionine is 322 amino acids in length and exhibits a molecular mass of 35,546. Comparison of the protein sequence to the sequences of the four members of the mitochondrial carrier protein family (ADP/ATP carrier, phosphate carrier, 2-oxoglutarate/malate carrier, and uncoupling protein) does not reveal significant similarity (cf. Walkeret al., 1987). A tripartite internal homology, which is a characteristic of these proteins, is not present in the sequence of the tricarboxylate carrier protein. The mRNA for the tricarboxylate carrier is expressed in rat liver and brain, but not in rat heart.     

Kinetic Evidence for the Uniport Mechanism Hypothesis in the Mitochondrial Tricarboxylate Transport System

The kinetics of the transport of citrate by the tricarboxylate transport system located in the inner mitochondrial membrane was studied in proteoliposomes containing the purified carrier protein, in order to verify the previously hypothesized mechanism of uniport (J. Bioenerg. Biomembr. 35, 133–140, 2003) and achieve some information on the kinetic properties of the carrier transport system. For this purpose, a mathematical model has been elaborated and the experimental data were analyzed according to it. The results indicate that the data actually fit with the uniport model, and hence it is confirmed that the carrier has a single binding site for its substrates and can oscillate between the inside and outside form, in both the free and substrate-bound states. The rearrangement of the free form is slower than the bound form in both directions. The dissociation constants for the internal substrate are at least one order of magnitude higher than the one for external citrate. As a consequence of these last two points, the rate of citrate transport by the carrier is much higher when it operates in exchange with another substrate than when it operates in net uniport.     

Identification and characterization of a novel mitochondrial tricarboxylate carrier

Here we report the identification and functional characterization of a novel mitochondrial tricarboxylate carrier protein, designated BBG-TCC, in rat brain. The cDNA encodes the predicted protein of 342-amino acid residues with five putative membrane-spanning domains. The protein has apparent similarity with a mitochondrial tricarboxylate carrier TCC, but is distinct from the other mitochondria anion transporters. BBG-TCC shows a citrate transport activity. It is specifically expressed in the brain and localizes in the mitochondria of Bergmann glial cells. In contrast, the expression of TCC is rather ubiquitous and strong in neuronal cells in the brain. This new family of proteins may contribute to biosynthesis and bioenergetics in the brain.     

Biogenesis of rat mitochondrial citrate carrier (CIC): the N-terminal presequence facilitates the solubility of the preprotein but does not act as a targeting signal

Most mitochondrial preproteins carry a cleavable N-terminal presequence that mediates targeting to mitochondria and translocation across the mitochondrial membranes. In this study, we characterized the presequence of the citrate carrier (CIC, tricarboxylate carrier) of rat liver mitochondria. The CIC presequence was found to be dispensable both for targeting to mitochondria and insertion into the inner membrane. Unlike the presequence of the related phosphate carrier, fusion of the CIC presequence to the cytosolic enzyme dihydrofolate reductase did not confer mitochondrial targeting, indicating that the CIC presequence does not act as a targeting signal. However, the presequence was required to keep the CIC in a soluble state. Mature CIC lacking the presequence was prone to aggregation. We conclude that mitochondrial presequences do not necessarily act as mediators of targeting. In the case of the CIC, the presequence appears to determine the folding state of the preprotein.     

High-level bacterial expression of mitochondrial transport proteins

The inability to obtain abundant quantities of purified, functional membrane proteins has represented a significant impediment to the goal of obtaining high-resolution structural information. In this review, procedures are described which have been developed in this laboratory and enable the high-level bacterial expression and subsequent purification of functional mitochondrial citrate transport proteins from yeast and rat liver. The data that we have obtained using these procedures and related results from other laboratories are discussed. Additionally, the general applicability of this approach to most mitochondrial transport proteins as well as to other types of membrane proteins is considered. Finally, relevant considerations when contemplating the use of this methodology and the likely value of this approach in future areas of research are explored.     

The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini

The mitochondrial tricarboxylate carrier plays a fundamental role in the hepatic fatty acid synthesis. In this study, we investigated the transmembrane organization of this protein in the inner membrane of eel liver mitochondria using anti–N-terminal and anti–C-terminal antibodies. These antibodies recognized the N- and C-termini of the tricarboxylate carrier in intact mitoplasts, thus suggesting a cytosolic exposure of these regions in the membrane-bound protein. This structural arrangement of the tricarboxylate carrier was further confirmed by protease treatment of intact mitoplasts. Moreover, the oligomeric state of the native tricarboxylate carrier was investigated by blue native electrophoresis. A dimeric form of the carrier protein was found when eel liver mitochondria were solubilized with the mild detergent digitonin. These findings suggest an arrangement of the dimeric tricarboxylate carrier into an even number of membrane-spanning domains, with the N-terminal and C-terminal regions oriented toward the intermembrane space of fish mitochondria.     

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.     

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers

The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.     

Structure,function and regulation of the tricarboxylate transport protein from rat liver mitochondria

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning -helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.     

Anion transport in rat brain mitochondria: Fumarate uptake via the dicarboxylate carrier

Penetration of fumarate into rat brain mitochondria has been investigated, as required in brain ammoniogenesis. Mitochondria swell in ammonium fumarate and this swelling is increased by both Pi and malate. According to a carrier mediated process, fumarate translocation, which occurs in exchange with intramitochondrial malate or Pi shows saturation characteristics. By photometrically investigating the kinetics of fumarate/malate, fumarate/ Pi and malate/Pi exchanges, different Km values were obtained (10, 22 and 250 M, respectively), whereas no significant difference was found forV _max values (40 nmol NAD(P)⁺ reduced/min×mg protein). This suggests that fumarate and malate share a single carrier to enter mitochondria, namely the dicarboxylate carrier. Both comparison made of theV _max values and inhibiton studies exclude a fumarate translocation via either the tricarboxylate carrier, whose occurrence in brain is here demonstrated, or oxodicarboxylate carrier. Kinetic investigation of the dicarboxylate translocator shows the existence of thiol group/s and metal ion/s at or near the substrate binding sites. The experimental findings are discussed in the light of fumarate uptake in vivo in brain ammoniogenesis.Abbreviations AD.SUCC adenylsuccinate - ASP aspartate - BTA 1,2,3,-benzenetricarboxylate - CITR citrate - D-NAD deamino-NAD - PUM fumarate - GABA -aminobutyrate - GAP glyceraldehyde-3-phosphate - GAP-DH glyceraldheyde-3-phosphate dehydrogenase - GHBA -hydroxybutyrate - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - OAA oxaloacetate - OG oxoglutarate - PEP phosphoenolpyruvate - 3-PG glycerate-3-phosphate - 3-PGP glycerate-1,3-diphosphate - PYR pyruvate - RBM rat brain mitochondria - RHM rat heart mitochondria - RKM rat kidney mitochondria - RLM rat liver mitochondria - SSA succinic semialdehyde     

Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1

The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.     

Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.     

Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: Effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide

Summary Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outwardfacing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971)J. Physiol. (London) 213:647–667; Edwards, P.A. (1973)Biochim. Biophys. Acta 311:123–140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since¹⁴C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inwardfacing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carriersubstrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.     

Cell-free synthesis, reconstitution, and characterization of a mitochondrial dicarboxylate-tricarboxylate carrier of Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, was recently shown to operate a branched pathway of tricarboxylic acid (TCA) metabolism. To identify and characterize membrane transporters required for such TCA metabolism in the parasite, we isolated a cDNA for a dicarboxylate–tricarboxylate carrier homolog (PfDTC), synthesized the encoded protein with the use of a cell-free translation system, and determined the substrate specificity of its transport activity with a proteoliposome reconstitution system. PfDTC was found to mediate efficient oxoglutarate–malate, oxoglutarate–oxaloacetate, or oxoglutarate–oxoglutarate exchange across the liposome membrane. Our results suggest that PfDTC may mediate the oxoglutarate–malate exchange across the inner mitochondrial membrane required for the branched pathway of TCA metabolism in the malaria parasite.     

Inhibition of mitochondrial substrate anion translocators by a synthetic amphipathic polyanion

A synthetic polyanion (a copolymer of methacrylate, maleate, and styrene in 1:2:3 proportion with an average molecular weight of 10,000 dalton) inhibits the tricarboxylate, oxoglutarate, dicarboxylate, and adenine nucleotide translocators of rat liver mitochondria. The activity versus inhibitor concentration curves are sigmoidal. The inhibition of the oxoglutarate and tricarboxylate translocators by the polyanion is competitive, while that of the adenine nucleotide translocator is of mixed-type. TheK ₁ values of the polyanion are the following: for oxoglutarate translocator 4.0 µM, tricarboxylate translocator 1.2 µM, and adenine nucleotide translocator 1.3 µM with ADP and 0.8 µM with ATP. It is suggested that the polyanion acts primarily by increasing the negative charge of the inner membrane at the outer surface, and the sensitivity of the translocators toward the polyanion depends on the number of negative charges of their substrates.     

Mechanism and regulation of the mitochondrial ATP-Mg/Pi carrier

The mitochondrial ATP-Mg/P_i carrier functions to modulate the matrix adenine nucleotide pool size (ATP + ADP + AMP). Micromolar Ca²⁺ is required to activate the carrier. Net adenine nucleotide transport occurs as an electroneutral divalent exchange of ATP-Mg^2– for HPO ₄ ^2– . A steady-state adenine nucleotide pool size is attained when the HPO ₄ ^2– and ATP-Mg^2– matrix/cytoplasm concentration ratios are the same. This means that ATP-Mg^2– can be accumulated against a concentration gradient in proportion to the [HPO ₄ ^2– ] gradient that is normally maintained by the P_i/OH^– carrier. In liver, changes in matrix adenine nucleotide concentrations that are brought about by the ATP-Mg/P_i carrier can affect the activity of adenine nucleotide-dependent enzymes that are in the mitochondrial compartment. These enzymes in turn contribute to the overall regulation of bioenergetic function, flux through the gluconeogenesis and urea synthesis pathways, and organelle biogenesis. The ATP-Mg/P_i carrier is distinct from other mitochondrial transport systems with respect to kinetics and to substrate and inhibitor sensitivity. It is the only carrier regulated by Ca²⁺. This carrier is present in kidney and liver mitochondria, but not in heart.     

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an α-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Natural and azido fatty acids inhibit phosphate transport and activate fatty acid anion uniport mediated by the mitochondrial phosphate carrier

The electroneutral P(i) uptake via the phosphate carrier (PIC) in rat liver and heart mitochondria is inhibited by fatty acids (FAs), by 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and heptylbenzoic acid ( approximately 1 microm doses) and by lauric, palmitic, or 12-azidododecanoic acids ( approximately 0.1 mm doses). In turn, reconstituted E. coli-expressed yeast PIC mediated anionic FA uniport with a similar pattern leading to FA cycling and H(+) uniport. The kinetics of P(i)/P(i) exchange on recombinant PIC in the presence of AzDA better corresponded to a competitive inhibition mechanism. Methanephosphonate was identified as a new PIC substrate. Decanephosphonate, butanephosphonate, 4-nitrophenylphosphate, and other P(i) analogs were not translocated and did not inhibit P(i) transport. However, methylenediphosphonate and iminodi(methylenephosphonate) inhibited both electroneutral P(i) uptake and FA cycling via PIC. AzDA analog 16-(4-azido-2-nitrophenylamino)-[(3)H(4)]-hexadecanoic acid ((3)H-AzHA) bound upon photoactivation to several mitochondrial proteins, including the 30- and 34-kDa bands. The latter was ascribed to PIC due to its specific elution pattern on Blue Sepharose and Affi-Gel. (3)H-AzHA photolabeling of recombinant PIC was prevented by methanephosphonate and diphosphonates and after premodification with 4-azido-2-nitrophenylphosphate. Hence, the demonstrated PIC interaction with monovalent long-chain FA anions, but with divalent phosphonates of short chain only, indicates a pattern distinct from that valid for the mitochondrial uncoupling protein-1.