Dicarboxylate Exchange Research Articles

Drosophila INDY and Mammalian INDY: Major Differences in Transport Mechanism and Structural Features despite Mostly Similar Biological Functions.

INDY (I’m Not Dead Yet) is a plasma membrane transporter for citrate, first identified in Drosophila. Partial deficiency of INDY extends lifespan in this organism in a manner similar to that of caloric restriction. The mammalian counterpart (NaCT/SLC13A5) also transports citrate. In mice, it is the total, not partial, absence of the transporter that leads to a metabolic phenotype similar to that caloric restriction; however, there is evidence for subtle neurological dysfunction. Loss-of-function mutations in SLC13A5 (solute carrier gene family 13, member A5) occur in humans, causing a recessive disease, with severe clinical symptoms manifested by neonatal seizures and marked disruption in neurological development. Though both Drosophila INDY and mammalian INDY transport citrate, the translocation mechanism differs, the former being a dicarboxylate exchanger for the influx of citrate2− in exchange for other dicarboxylates, and the latter being a Na+-coupled uniporter for citrate2−. Their structures also differ as evident from only ~35% identity in amino acid sequence and from theoretically modeled 3D structures. The varied biological consequences of INDY deficiency across species, with the beneficial effects predominating in lower organisms and detrimental effects overwhelming in higher organisms, are probably reflective of species-specific differences in tissue expression and also in relative contribution of extracellular citrate to metabolic pathways in different tissues

Malate valves: old shuttles with new perspectives.

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyse the reversible interconversion of malate and oxaloacetate and their transport. Depending on the co‐enzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes. Activities of NAD‐dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids. In addition, chloroplasts possess a NADP‐dependent MDH isoform. The NADP‐MDH as part of the ‘light malate valve’ plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post‐translational redox‐modification mediated via the ferredoxin‐thioredoxin system and fine control via the NADP +/NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD‐MDH (‘dark malate valve’) is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, knowledge about regulation of the other two cytosolic MDHs as well as NAD‐MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria and peroxisomes have been characterised, but not much is known about cytosolic NAD‐MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange, focusing on the various metabolic functions of these valves.

Role of Mouse Organic Anion Transporter 3 (mOat3) as a Basolateral Prostaglandin E2 Transport Pathway

Renal organic anion transporters play an important role in the handling of a number of endogenous and exogenous anionic substances in the kidney. In this study, we investigated prostaglandin E2 (PGE2) transport properties and intrarenal localization of mouse organic anion transporter 3 (mOat3). When expressed in Xenopus oocytes, mOat3 mediated the time- and concentration-dependent transport of PGE2 (Km: 1.48 µM). PGE2 transport mediated by mOat3 was trans-stimulated by intracellular glutarate injected into the oocytes. PGE2 efflux via mOat3 was also trans-stimulated by extracellular glutarate. Thus, mOat3 was shown to mediate the bidirectional transport of PGE2, partly coupled to the dicarboxylate exchange mechanism. Immunohistochemical study revealed that mOat3 protein was localized at the basolateral membrane of renal proximal and distal tubules. Furthermore, diffuse expression of mOat3, including expression in the basolateral membrane in macula densa (MD) cells, was observed. These results indicate that mOat3 plays an important role as a basolateral transport pathway of PGE2 in the distal nephron including MD cells that may constitute one of the indispensable steps for renin release and regulation of the tubuloglomerular feedback mechanism.

Characterization of a dicarboxylate exchange system able to exchange pyrophosphate for l-malate in non-photosynthetic plastids from developing maize embryos

Non-photosynthetic plastids from plant tissues are unable to fix carbon dioxide into carbohydrates, and so, they depend on carriers of the inner plastid membrane. Spinach chloroplasts are known to have a dicarboxylate translocator (DL) system able to exchange l-malate for other dicarboxylates. This paper presents evidence for the existence of a similar, but not equal system in non-photosynthetic plastids, isolated from developing maize embryos. Plastids were isolated by Percoll gradient centrifugation. Dicarboxylate uptake or release was determined by centrifugation through a single silicone layer. We found a dicarboxylate translocating activity able to exchange l-malate, for either succinate, glutamate, oxaloacetate, 2-oxoglutarate or aspartate, and also malonate and the tricarboxylate, citrate. Surprisingly, this activity also exchanged malate for pyrophosphate and in addition, pyrophosphate inhibited the dicarboxylate exchange. We found a competitive inhibition pattern of the l-malate/malonate exchange by n-butylmalonate, and this compound also inhibited the l-malate/pyrophosphate exchange. Our results are consistent with the participation of, at least, one common component in the l-malate/malonate and l-malate/pyrophosphate carrier systems. We discuss the possible role of the dicarboxylate exchange in the plastidial and cytoplasm metabolism in developing maize embryos.

Mouse reduced in osteosclerosis transporter functions as an organic anion transporter 3 and is localized at abluminal membrane of blood-brain barrier.

The "reduced in osteosclerosis" transporter (Roct), which shows decreased expression in the osteosclerosis (oc) mutant mouse, has high homology with rat and human organic anion transporter 3 (OAT3). However, its transport properties and involvement in bone turnover are poorly understood. Here, we examined Roct-mediated transport using a Xenopus laevis oocyte expression system. Roct-expressing oocytes exhibited uptake of [(3)H]estrone sulfate, [(3)H]p-aminohippuric acid, [(3)H]benzylpenicillin, [(3)H]estradiol 17beta-glucronide, [(3)H]indoxyl sulfate, [(14)C]indomethacin, [(3)H]homovanillic acid, [(3)H]cimetidine, [(14)C]glutarate, [(14)C]salicylic acid, and [(3)H]methotrexate. Furthermore, the uptake of [(3)H]benzylpenicillin by Roct coexpressed with Na(+)-dicarboxylate cotransporter was trans-stimulated by glutarate preloading, and [(3)H]estrone sulfate uptake showed a similar tendency, suggesting that Roct is a dicarboxylate exchanger. [(3)H]Benzylpenicillin uptake by Roct was inhibited by OAT3 substrates and inhibitors, and by sulfate or glucuronide conjugates, and compounds involved in bone turnover. Roct mRNA is expressed abundantly in the kidney and was also detected in the brain, choroid plexus, and eye. Immunohistochemical analysis revealed that Roct is localized in brain capillary endothelial cells. These results indicate that the transport properties and tissue distribution of Roct are similar to those of OAT3, suggesting that Roct functions as mouse OAT3. Because Roct is expressed in the kidney and at the blood-brain barrier, it may play a role in the excretion of substrates such as conjugates and bone turnover factors.

Human Organic Anion Transporter 3 (hOAT3) can Operate as an Exchanger and Mediate Secretory Urate Flux

Background/Aims: Renal secretion of organic anions is critically dependent on their basolateral uptake against the electrochemical gradient. Due to their localization, two transporters are likely involved, namely OAT1 and OAT3. While OAT1 as an exchanger clearly operates in the secretory direction, OAT3 in its previously supposed mode as a uniporter should move anionic substrates from cell to blood. It would thus dissipate gradients established by OAT1 of common OAT1/OAT3 substrates. In the present study we therefore reinvestigated the driving forces of human OAT3. Methods: The human OAT3 obtained from the Resource Center/Primary Database was made functional by site-directed mutagenesis. Using the Xenopus laevis oocyte expression system, hOAT3-mediated transport of estrone sulfate (ES) and dicarboxylates was assayed for cis-inhibition and/or trans-stimulation in both the uptake and efflux direction. Results: hOAT3-mediated efflux of glutarate (GA), can be significantly trans-stimulated by a variety of ions with high cis-inhibitory potency, including GA (282%), α-ketoglutarate (476%), p-aminohippurate (179%), and, most notably, urate (167%). Urate cis-inhibited ES uptake with an IC₅₀ close to normal serum urate concentrations. Conclusion: These data indicate that OAT3 does not represent a uniporter but operates as an organic ion%dicarboxylate exchanger similar to OAT1, and may mediate renal urate secretion.

Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient.

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na(+) gradient through Na(+)-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na(+)-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li(+) or methylsuccinate (MS) or when Na(+) was absent. All these treatments inhibit the Na(+)-dicarboxylate cotransporter, but not rat Oat3. Li(+), MS, and Na(+) removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na(+) sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li(+), MS, taurocholate, and removal of Na(+). Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na(+) gradient.

Basolateral transport of glutarate in proximal S2 segments of rabbit kidney: kinetics of the uptake process and effect of activators of protein kinase A and C.

The kinetics of tubular glutarate uptake, the coupling of glutarate to p-aminohippurate (PAH) transport and the effect of activators of protein kinase A and C on glutarate uptake were studied using isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of [14C]glutarate reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s glutarate uptake measurements were performed. In a first set of experiments it could be shown that preloading proximal S2 segments with glutarate (10(-3 )M) stimulated [3H]PAH uptake indicating that glutarate may be a substrate of the PAH /dicarboxylate exchanger. The kinetic data revealed a Km value of 0. 62 mM and a Vmax value of 84.1 pmol nl-1min-1 for tubular [14C]glutarate uptake across the basolateral cell membrane. In contrast to basolateral PAH transport (previous studies from this laboratory), tubular 30 s [14C]glutarate uptake was not affected by either the phorbol ester phorbol 12-myristate 13-acetate (PMA, 10(-7 )M), an activator of protein kinase C, or by the membrane-permeant analogues of cAMP, dibutyryl cyclic AMP (db-cAMP, 10(-4 )M) and 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP, 10(-4 )M). The results indicate that the protein kinases A and C have no function in the regulation of the renal basolateral dicarboxylate transporter. This finding agrees well with the structural feature of the recently cloned rabbit renal dicarboxylate transporter which does not contain any putative phosphorylation sites for protein kinase C or cAMP-dependent kinase.

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.

Transport of Dicarboxylic Acids in Castor Bean Mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of (14)C from [(14)C]malate or [(14)C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of (14)C from [(14)C]malate or [(14)C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K(m) values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [(14)C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [(14)C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4 degrees C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [(14)C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K(m) of 2.5 millimolar and V(max) of 250 nanomoles per minute per milligram protein at 4 degrees C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

Reconstitution of the dicarboxylate exchange activity by incorporation into liposomes of a Triton-extract of mitochondrial rat-liver inner membranes

The exchange between external [ 14C] malonate and internal malate or phosphate was reconstituted in liposomes prepared by incorporation of a Triton-extract of mitochondrial rat-liver inner membranes. The conditions of transport were investigated and the kinetic parameters of malonate-malate and malonate-phosphate exchanges were determined. The exchange was sensitive to butylmalonate and to organomercurials. Sulfate and phosphate decreased the rate of malonatemalate exchange and phosphate inhibition was found to be competitive. This report demonstrates the possibility to reconstitute a functional dicarboxylate transport and to use the system for further purification and for studies at the molecular level.

Nitrate Reduction by a Dicarboxylate Shuttle in a Reconstituted System from Spinach Leaves

A reconstituted system of supernatant and mitochondria from spinach leaves reduced NO3- in the presence of NAD+. A faster rate (2-6 times) of NO3- reduction was obtained when oxaloacetate (OAA) and glycine were present. The reduction of NO3- in the presence of OAA and glycine was sensitive to inhibition by n-butyl malonate, suggesting that the dicarboxylate exchange carrier was involved in the transport of malate out of the mitochondria. The capacity of the dicarboxylate shuttle to reduce NO,- was 63 % that of the in vitvo nitrate reductase activity in the supernatant

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained. It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μ m palmitate, palmitoyl-CoA, or palmitoyl- l-carnitine. At palmitoyl-CoA concentrations higher than 30 μ m oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO 2. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of 14CO 2 and 14C-labeled perchloric acid-soluble products is higher from [1- 14C]palmitate than that from [ U- 14C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of 14CO 2 production and 14C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring 14C-fatty acid oxidation than that of 14CO 2 production alone.

On the mechanism of phosphate and dicarboxylate transport in mitochondria

Studies on the swelling of mitochondria in isoosmotic solutions of the ammonium salts of phosphoric acid and of various dicarboxylic and tricarboxylic acids, led to the postulation of 3 functionallyrelated but separate anion transporters in the inner mitochondrial membrane [ 1,2] : a phosphate/OH-, a phosphate/dicarboxylate and a dicarboxylate/tricarboxylate antiporter. In further studies [3-61, using the swelling technique [7-91, or more direct methods to study the transport of anions [lo-l 51, the main conclusions were further substantiated (reviewed [17-211). Although the 2 transport systems for phosphate could be distinguished by the discovery of the specific inhibitors N-ethylmaleimide for phosphate/OHexchange [5,1 l] and butylmalonate for phosphate/ dicarboxylate exchange [3 ,l 1 ] , experiments on the effect of the 2 inhibitors on mitochondrial phosphate and dicarboxylate exchanges, led to the proposition that the inner mitochondrial membrane contains only 1 carrier for phosphate and dicarboxylates [22,23] . Kinetic analysis by a modified Hill-plot of the effect of N-ethylmaleimide and butylmalonate on 32P uptake in m’ o rt chondria clearly demonstrated a mutual interdependence of both inhibitor binding sites [24]. in an investigation on the induction of mitochondrial swelling in NH4 -malate by the phosphate analogues thiophosphate, monoand di-fluorophosphate, we concluded that the uptake of malate into mitochondria does not occur via an exchange with phosphate, but by activation of the transporter by phosphate [ 251.

The mitochondrial pyruvate carrier, its exchange properties and its regulation by glucagon

1. Introduction Earlier reports [ 1,2] have demonstrated the presence of a pyruvate carrier in rat liver mitochondria. The evidence was pH dependence of uptake and efflux of pyruvate suggesting exchange with hydroxide anions, saturation kinetics and the existence of a specific inhibitor, cw-cyano4-hydroxy-cinnamic acid. It was reported that phosphate and citrate anions would not exchange for pyruvate [l] but later there has been disagreement as to whether dicarboxylate anions could exchange with pyruvate across fhe mitochondrial membrane (cf. [3,4] ). Because of this and because of the potential relevance of pyruvate/ dicarboxylate exchange to the regulation of gluconeo- genesis we have reinvestigated this point using a recently published technique [S] . This technique, which uses cu-cyano-3-hydroxy-cinnamic acid as an ‘inhibitor-stop’, has the merit (in comparison with other methods) of measuring initial rates of uptake along a controlled pH gradient at temperatures near to physiological. Our results suggest that such a pyruvate/dicarboxylate exchange does occur and that it probably involves the pyruvate carrier only. Using the same technique we have also investigated the possibility suggested by others [6-81 that glucagon stimulation of gluconeogenesis in the liver in situ or in liver cells incubated in vitro may involve stimulation of pyruvate entry into the mitochondria. The suggestion was based on indirect evidence, namely increased rates of pyruvate decarboxylation and carboxylation by isolated mitochondria from liver exposed to high concentrations of glucagon. We have no shown directly that there is in fact a con-

Ion transport by heart mitochondria: XXVI. Carrier-mediated anion transport by isolated beef heart mitochondria

The uptake and release of P i and substrate anions has been examined in isolated beef heart mitochondria. Evidence is presented that these mitochondria like those from rat liver, contain a P i OH − exchanger which is sensitive to mercurials and to N-ethylmaleimide and a dicarboxylate exchanger which is capable of exchanging P i for P i, P i for dicarboxylate, and dicarboxylate for dicarboxylate. Exchange on this carrier is inhibited by mercurials and the ability of this transporter to catalyze P i for dicarboxylate exchange in either direction is quantitatively much less than is found in liver mitochondria. It is concluded that previous indications of differences in divalent anion uptake between liver and heart mitochondria are the result of this quantitative discrepancy and do not indicate that fundamental differences in anion uptake mechanism are in operation. Heart mitochondria also contain an α-keto-glutarate exchanger and a tricarboxylate exchanger with properties analogous to the transporters described for other mitochondria, but with quantitative differences apparent in various measuring systems. These studies agree, for the most part, with the conclusions of Sluse, Meijer, and Tager ( Fed. Eur. Biochem. Soc. Lett. 18, 149, 1971) using rat heart mitochondria.