Ascorbate Transporter Research Articles

Context-dependent Cryptic Roles of Specific Residues in Substrate Selectivity of the UapA Purine Transporter

Members of the ubiquitous Nucleobase Ascorbate Transporter (NAT) family are H+ or Na+ symporters specific for the cellular uptake of either purines and pyrimidines or L-ascorbic acid. Despite the fact that several bacterial and fungal members have been extensively characterised at a genetic, biochemical or cellular level, and crystal structures of NAT members from Escherichia coli and Aspergillus nidulans have been determined pointing to a mechanism of transport, we have little insight on how substrate selectivity is determined. Here, we present systematic mutational analyses, rational combination of mutations, and novel genetic screens that reveal cryptic context-dependent roles of partially conserved residues in the so-called NAT signature motif in determining the specificity of the UapA transporter of A. nidulans. We show that specific NAT signature motif substitutions, alone and in combinations with each other or with distant mutations in residues known to affect substrate selectivity, lead to novel UapA versions possessing variable transport capacities and specificities for nucleobases. In particular, we show that a UapA version including the quadruple mutation T405S/F406Y/A407S/Q408E in the NAT signature motif (UapA-SYSE) becomes incapable of purine transport, but gains a novel pyrimidine-related profile, which can be further altered to a more promiscuous purine/pyrimidine profile when combined with replacements at distantly located residues, especially at F528. Our results reveal that UapA specificity is genetically highly modifiable and allow us to speculate on how the elevator-type mechanism of transport might account for this flexibility.

Activation of adenosine A3 receptors regulates vitamin C transport and redox balance in neurons

Adenosine is an important neuromodulator in the CNS, regulating neuronal survival and synaptic transmission. The antioxidant ascorbate (the reduced form of vitamin C) is concentrated in CNS neurons through a sodium-dependent transporter named SVCT2 and participates in several CNS processes, for instance, the regulation of glutamate receptors functioning and the synthesis of neuromodulators. Here we studied the interplay between the adenosinergic system and ascorbate transport in neurons. We found that selective activation of A3, but not of A1 or A2a, adenosine receptors modulated ascorbate transport, decreasing intracellular ascorbate content. Förster resonance energy transfer (FRET) analyses showed that A3 receptors associate with the ascorbate transporter SVCT2, suggesting tight signaling compartmentalization between A3 receptors and SVCT2. The activation of A3 receptors increased ascorbate release in an SVCT2-dependent manner, which largely altered the neuronal redox status without interfering with cell death, glycolytic metabolism, and bioenergetics. Overall, by regulating vitamin C transport, the adenosinergic system (via activation of A3 receptors) can regulate ascorbate bioavailability and control the redox balance in neurons.

Experimental evidence and physiological significance of the ascorbate passive diffusion through the lipid bilayer

The diverse range of functions performed by ascorbate in many metabolic processes requires its effective redistribution between various aqueous body compartments. It is believed that this hydrophilic molecule needs protein transporters for crossing the biological membrane barriers. Any effective model reflecting the ascorbate distribution within the body requires bi-directional fluxes, but only the ascorbate transporters facilitating its intake by cells have been identified to date. The cellular efflux of this molecule still lacks proper mechanistic explanation, nevertheless data suggesting possible passive ascorbate transport recently appeared. In the paper, we provide experimental evidences that ascorbate associates efficiently with the lipid bilayer interface and slowly crosses its hydrophobic core. The measured logPmembrane/water and membrane permeability coefficient equal to 3 and 10−7 – 10−8 cm/s, respectively. The ascorbate passive diffusion across the lipid bilayer provides the missing element needed for the construction of a consistent physiological model describing the ascorbate local homeostasis. The model was effectively used for the construction of the mechanistic description of the processes, which facilitate the ascorbate homeostasis in the brain.

Metabolic Regulation of Stem Cell Fate and Function

The overarching goal of this project is to understand how diet‐derived nutrients regulate stem cell metabolism and downstream fate decisions in the setting of normal development and disease. Quiescent adult stem cells typically favor glycolytic metabolism over the oxidative metabolism utilized by differentiating cells. Importantly, manipulating stem cell metabolism is sufficient to alter cell fate decisions. While the serum concentrations of endogenous metabolites such as glucose and lactate are under homeostatic constraints, diet can cause vast fluctuations in circulating levels of certain metabolites that serve as co‐factors for chromatin and DNA modifying enzymes. As such, exogenous nutrients can control the epigenome and gene expression programs, thus altering differentiation and cell fate. However, little is known about how changing nutrient levels will affect the metabolism and chromatin status in the adult stem cell populations. To address this question, we have adapted a recently described mouse liver organoid system. The liver coordinates whole‐body metabolism and detoxification, and therefore is acutely tuned to respond to dietary changes. Liver organoids generate 3D assemblies that mirror the native physiology of the tissue and self‐renew in vitro due to the activity of Wnt‐responsive stem cells that reside in the hepatic ductal compartment. We have found that treatment with physiologically attainable concentrations of ascorbic acid inhibits hepatic organoid expansion in a dose‐dependent manner. Ascorbic acid, commonly known as Vitamin C, is a water‐soluble antioxidant that is obtained exclusively through the diet in humans. Ascorbic acid is an essential co‐factor for the Fe‐ and α‐ketoglutarate dependent dioxygenase (α‐KGDDs) class of enzymes, which includes collagen hydroxylases, prolyl hydroxylases, and epigenetic erasers such as DNA and histone demethylases. To determine if activation of α‐KGDDs enzymes suppresses organoid expansion, we treated hepatic organoids with a cell permeable form of α‐KG and observed growth inhibition identical to that observed with ascorbic acid treatment. α‐KG/succinate ratios and expression of the ascorbate transporters increases in differentiated hepatic organoids compared to cultures maintained in expansion media, suggesting that ascorbic acid may preferentially accumulate and function during terminal differentiation. Taken together, we propose that ascorbic acid acts as a cofactor for α‐KGDD enzymes in order to restrain stem cell activity and may play a role in promoting differentiation to the hepatic lineage.Support or Funding InformationS.N.S is supported by a postdoctoral grant from the Eli and Edythe Broad Stem Cell Research Center at UCLA. H.R.C is supported by a CTSI seed grant from the David Geffen School of Medicine at UCLA.

Vitamin C in Plants: Novel Concepts, New Perspectives, and Outstanding Issues.

Significance: The concept that vitamin C (l-ascorbic acid) is at the heart of the peroxide processing and redox signaling hub in plants is well established, but our knowledge of the precise mechanisms involved remains patchy at best. Recent Advances: Ascorbate participates in the multifaceted signaling pathways initiated by both reactive oxygen species (ROS) and reactive nitrogen species. Crucially, the apoplastic ascorbate/dehydroascorbate (DHA) ratio that is regulated by ascorbate oxidase (AO) sculpts the apoplastic ROS (apoROS) signal that controls polarized cell growth, biotic and abiotic defences, and cell to cell signaling, as well as exerting control over the light-dependent regulation of photosynthesis. Critical Issues: Here we re-evaluate the roles of ascorbate in photosynthesis and other processes, addressing the question of how much we really know about the regulation of ascorbate homeostasis and its functions in plants, or how AO is regulated to modulate apoROS signals. Future Directions: The role of microRNAs in the regulation of AO activity in relation to stress perception and signaling must be resolved. Similarly, the molecular characterization of ascorbate transporters and mechanistic links between photosynthetic and respiratory electron transport and ascorbate synthesis/homeostasis are a prerequisite to understanding ascorbate homeostasis and function. Similarly, there is little in vivo evidence for ascorbate functions as an enzyme cofactor.

Specific Residues in a Purine Transporter Are Critical for Dimerization, ER Exit, and Function.

Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

Identification of a Novel Nucleobase-Ascorbate Transporter Family Member in Fish and Amphibians

Nucleobase-Ascorbate Transporter (NAT) family includes ascorbic acid, nucleobases, and uric acid transporters: With broad evolutionary distribution. In vertebrates, four members have been previously recognized, the ascorbate transporters Slc23a1 and Slc3a2, the nucleobase transporter Slc23a4 and an orphan transporter Slc23a3. Using phylogenetic and synteny analysis, we identify a fifth member of the vertebrate slc23 complement (slc23a5), present in neopterygians (gars and teleosts) and amphibians, and clarify the evolutionary relationships between the novel gene and known slc23 genes. Further comparative analysis puts forward uric acid as the preferred substrate for Slc23a5. Gene expression quantification, using available transcriptomic data, suggests kidney and testis as major expression sites in Xenopus tropicalis (western clawed frog) and Danio rerio (zebrafish). Additional expression in brain was detected in D. rerio, while in the Neoteleostei Oryzias latipes (medaka) slc23a5 expression is restricted to the brain. The biological relevance of the retention of an extra transporter in fish and amphibians is discussed.

Transporter oligomerisation: roles in structure and function.

Oligomerisation is a key feature of integral membrane transporters with roles in structure, function and stability. In this review, we cover some very recent advances in our understanding of how oligomerisation affects these key transporter features, with emphasis on a few groups of transporters, including the nucleobase ascorbate transporters, neurotransmitter sodium symporters and major facilitator superfamily members.

Conjugation to Ascorbic Acid Enhances Brain Availability of Losartan Carboxylic Acid and Protects Against Parkinsonism in Rats.

Identification of renin-angiotensin system in the interplay of hypertension and neurodegeneration has paved the way for the repurposing of antihypertensive drugs against Parkinsonism. Losartan carboxylic acid (LCA), the potent AT1 blocker metabolite of losartan, suffers from poor bioavailability and brain access. Since ascorbate transporters have earlier shown enough flexibility as carriers, we have conjugated losartan carboxylic acid to ascorbic acid with the aim of achieving higher oral/brain availability. Ester of LCA and ascorbic acid (FED) was developed keeping in view the substrate specificity of ascorbate transporters. Oral/brain bioavailability was assessed using in vivo pharmacokinetic model. Effect on central nervous system (CNS) and protection against Parkinsonism was evaluated using in vivo models. FED enhanced bioavailability of LCA. The higher brain availability of LCA enabled CNS protection as evident from the increase in locomotor activity, improved motor coordination, and protection against drug-induced catatonia. In conclusion, FED offers an approach to repurpose LCA against Parkinsonism. This can encourage further investigation to simultaneously address hypertension and neurodegeneration.

Characterization of a Novel Mitochondrial Ascorbate Transporter From Rat Liver and Potato Mitochondria.

The Mitochondrial Ascorbic Acid Transporter (MAT) from both rat liver and potato mitochondria has been reconstituted in proteoliposomes. The protein has a molecular mass in the range of 28–35 kDa and catalyzes saturable, temperature and pH dependent, unidirectional ascorbic acid transport. The transport activity is sodium independent and it is optimal at acidic pH values. It is stimulated by proton gradient, thus supporting that ascorbate is symported with H+. It is efficiently inhibited by the lysine reagent pyridoxal phosphate and it is not affected by inhibitors of other recognized plasma and mitochondrial membranes ascorbate transporters GLUT1(glucose transporter-1) or SVCT2 (sodium-dependent vitamin C transporter-2). Rat protein catalyzes a cooperative ascorbate transport, being involved two binding sites; the measured K0.5 is 1.5 mM. Taking into account the experimental results we propose that the reconstituted ascorbate transporter is not a GLUT or SVCT, since it shows different biochemical features. Data of potato transporter overlap the mammalian ones, except for the kinetic parameters non-experimentally measurable, thus supporting the MAT in plants fulfills the same transport role.

Evolution of substrate specificity in the Nucleobase-Ascorbate Transporter (NAT) protein family.

L-ascorbic acid (vitamin C) is an essential metabolite in animals and plants due to its role as an enzyme co-factor and antioxidant activity. In most eukaryotic organisms, L-ascorbate is biosynthesized enzymatically, but in several major groups, including the primate suborder Haplorhini, this ability is lost due to gene truncations in the gene coding for L-gulonolactone oxidase. Specific ascorbate transporters (SVCTs) have been characterized only in mammals and shown to be essential for life. These belong to an extensively studied transporter family, called Nucleobase-Ascorbate Transporters (NAT). The prototypic member of this family, and one of the most extensively studied eukaryotic transporters, is UapA, a uric acid-xanthine/H+ symporter in the fungus Aspergillus nidulans. Here, we investigate molecular aspects of NAT substrate specificity and address the evolution of ascorbate transporters apparently from ancestral nucleobase transporters. We present a phylogenetic analysis, identifying a distinct NAT clade that includes all known L-ascorbate transporters. This clade includes homologues only from vertebrates, and has no members in non-vertebrate or microbial eukaryotes, plants or prokaryotes. Additionally, we identify within the substrate-binding site of NATs a differentially conserved motif, which we propose is critical for nucleobase versus ascorbate recognition. This conclusion is supported by the amino acid composition of this motif in distinct phylogenetic clades and mutational analysis in the UapA transporter. Together with evidence obtained herein that UapA can recognize with extremely low affinity L-ascorbate, our results support that ascorbate-specific NATs evolved by optimization of a sub-function of ancestral nucleobase transporters.

植物のアスコルビン酸輸送体がついに同定された : PHT4;4による葉緑体へのアスコルビン酸輸送

植物のアスコルビン酸輸送体がついに同定された : PHT4;4による葉緑体へのアスコルビン酸輸送

Compartment-Specific Importance of Ascorbate During Environmental Stress in Plants.

Ascorbate is an essential antioxidant in plants. Total contents and its redox state in organelles are crucial to fight and signal oxidative stress. Recent Advances: With quantitative immunoelectron microscopy and biochemical methods, highest ascorbate contents have recently been measured in peroxisomes (23 mM) and the cytosol (22 mM), lowest ones in vacuoles (2 mM), and intermediate concentrations (4-16 mM) in all other organelles. The accumulation of ascorbate in chloroplasts and peroxisomes is crucial for plant defense. Its depletion in chloroplasts, peroxisomes, and mitochondria during biotic stress leads to the accumulation of reactive oxygen species (ROS) and the development of chlorosis and necrosis. In the apoplast and vacuoles, ascorbate is the most important antioxidant for the detoxification of ROS. The cytosol acts as a hub for ascorbate metabolism as it reduces its oxidized forms that are produced in the cytosol or imported from other cell compartments. It is a sink for ascorbate that is produced in mitochondria, distributes ascorbate to all organelles, and uses ascorbate to detoxify ROS. As ascorbate and its redox state are involved in protein synthesis and modifications, it can be concluded that ascorbate in the cytosol senses oxidative stress and regulates plant growth, development, and defense. Future research should focus on (1) dissecting roles of ascorbate in vacuoles and the lumen of the endoplasmic reticulum, (2) identifying the physiological relevance of ascorbate transporters, and (3) correlating current data with changes in the subcellular distribution of related enzymes, ROS, and gene expression patterns.

Transporter oligomerization: form and function.

Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. Many transporters exist as oligomers in the membrane. Depending on the individual transport protein, oligomerization can have roles in membrane trafficking, function, regulation and turnover. For example, our recent studies on UapA, a nucleobase ascorbate transporter, from Aspergillus nidulans, have revealed both that dimerization of this protein is essential for correct trafficking to the membrane and the structural basis of how one UapA protomer can affect the function of the closely associated adjacent protomer. Here, we review the roles of oligomerization in many particularly well-studied transporters and transporter families.

Comprehensive genomic identification and expression analysis of the nucleobase-ascorbate transporter (NAT) gene family in apple

Nucleobase ascorbate transporters (NATs), also known as nucleobase:cation-symporter 2 (NCS2) proteins, belong to an evolutionarily widespread family of transport proteins. Because little is known about NAT genes in apple (Malus domestica), this papper screened GDR genome databases and identified 14 NATs in that species. Here, presented an overview of this gene family in apple, including structures, chromosome distribution and localization, phylogenies, and motifs. Phylogenetic and structure analyses showed that these genes could be assigned to three clusters. The 14 genes selected here displayed differential expression patterns in various tissues. Expression was very high for MdNAT12, MdNAT4, and MdNAT6 in the shoot tips, mature leaves, and young and mature fruits. MdNAT1 and MdNAT7 tended to show increased expression while the fruits were maturing. MdNAT6 proved responsive to both drought and salinity. Their transcript levels were generally up-regulated significantly during the fruit maturation process as well as in response to the imposition of drought or salt stress. Results suggest that members of this family function in tissue development, fruit maturation, and plant adaptations to adverse growing environments.

Ascorbic acid efflux from human brain microvascular pericytes: role of re-uptake.

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8-1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 H. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25-50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism.

Identification of the Elusive Chloroplast Ascorbate Transporter Extends the Substrate Specificity of the PHT Family

Most of the enzymatic reactions of ascorbate biosynthesis occur in the cytosol, with the exception of the terminal step, catalyzed by L-galactono-1,4-lactone dehydrogenase, which is localized on the inner mitochondrial membrane. Given the location of L-galactono-1,4-lactone dehydrogenase, the rate of ascorbate biosynthesis is intimately associated with the rate of mitochondrial respiration (Nunes-Nesi et al., 2005). Ascorbate has legion roles in the plant cell including mitosis, cell division, expansion and cell wall hydroxylation, hormone biosynthesis, signal transduction, programmed cell death, as well as protection against environmental stresses via detoxification of reactive oxygen species and regeneration of other antioxidants.

Ascorbic acid transport in brain microvascular pericytes

Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 min and an apparent transport Km of 21 μM, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion.

AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl−-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

Patterns of evolutionary conservation of ascorbic acid-related genes following whole-genome triplication in Brassica rapa.

Ascorbic acid (AsA) is an important antioxidant in plants and an essential vitamin for humans. Extending the study of AsA-related genes from Arabidopsis thaliana to Brassica rapa could shed light on the evolution of AsA in plants and inform crop breeding. In this study, we conducted whole-genome annotation, molecular-evolution and gene-expression analyses of all known AsA-related genes in B. rapa. The nucleobase–ascorbate transporter (NAT) gene family and AsA l-galactose pathway genes were also compared among plant species. Four important insights gained are that: 1) 102 AsA-related gene were identified in B. rapa and they mainly diverged 12–18 Ma accompanied by the Brassica-specific genome triplication event; 2) during their evolution, these AsA-related genes were preferentially retained, consistent with the gene dosage hypothesis; 3) the putative proteins were highly conserved, but their expression patterns varied; and 4) although the number of AsA-related genes is higher in B. rapa than in A. thaliana, the AsA contents and the numbers of expressed genes in leaves of both species are similar, the genes that are not generally expressed may serve as substitutes during emergencies. In summary, this study provides genome-wide insights into evolutionary history and mechanisms of AsA-related genes following whole-genome triplication in B. rapa.