Nucleobase-ascorbate Transporter Research Articles

The dimer of human SVCT1 is key for transport function

Humans and other primates lack the ability to synthesize the essential nutrient, Vitamin C, which is derived exclusively from the diet. Crucial for effective vitamin C uptake are the Na+ dependent Vitamin C transporters, SVCT1 and SVCT2, members of the nucleobase ascorbate transporter (NAT) family. SVCT1 and 2 actively transport the reduced form of Vitamin C, ascorbic acid, into key tissues. The recent structure of the mouse SVCT1 revealed the molecular basis of substrate binding and that, like the other structurally characterised members of the NAT family, it exists as a closely associated dimer. SVCT1 is likely to function via the elevator mechanism with the core domain of each protomer able to bind substrate and move through the membrane carrying the substrate across the membrane. Here we explored the function of a range of variants of the human SVCT1, revealing a range of residues involved in substrate selection and binding, and confirming the importance of the C-terminus in membrane localisation. Furthermore, using a dominant negative mutant we show that the dimer is essential for transport function, as previously seen in the fungal homologue, UapA. In addition, we show that a localisation deficient C-terminal truncation of SVCT1 blocks correct localisation of co-expressed, associated wildtype SVCT1. These results clearly show the importance of the dimer in both correct SVCT1 trafficking and transport activity.

Structures and mechanisms of the Arabidopsis cytokinin transporter AZG1.

Cytokinins are essential for plant growth and development, and their tissue distributions are regulated by transmembrane transport. Recent studies have revealed that members of the 'Aza-Guanine Resistant' (AZG) protein family from Arabidopsis thaliana can mediate cytokinin uptake in roots. Here we present 2.7 to 3.3 Å cryo-electron microscopy structures of Arabidopsis AZG1 in the apo state and in complex with its substrates trans-zeatin (tZ), 6-benzyleaminopurine (6-BAP) or kinetin. AZG1 forms a homodimer and each subunit shares a similar topology and domain arrangement with the proteins of the nucleobase/ascorbate transporter (NAT) family. These structures, along with functional analyses, reveal the molecular basis for cytokinin recognition. Comparison of the AZG1 structures determined in inward-facing conformations and predicted by AlphaFold2 in the occluded conformation allowed us to propose that AZG1 may carry cytokinins across the membrane through an elevator mechanism.

Vitamin C transporter SVCT1 serves a physiological role as a urate importer: functional analyses and in vivo investigations

Uric acid, the end product of purine metabolism in humans, is crucial because of its anti-oxidant activity and a causal relationship with hyperuricemia and gout. Several physiologically important urate transporters regulate this water-soluble metabolite in the human body; however, the existence of latent transporters has been suggested in the literature. We focused on the Escherichia coli urate transporter YgfU, a nucleobase-ascorbate transporter (NAT) family member, to address this issue. Only SLC23A proteins are members of the NAT family in humans. Based on the amino acid sequence similarity to YgfU, we hypothesized that SLC23A1, also known as sodium-dependent vitamin C transporter 1 (SVCT1), might be a urate transporter. First, we identified human SVCT1 and mouse Svct1 as sodium-dependent low-affinity/high-capacity urate transporters using mammalian cell-based transport assays. Next, using the CRISPR-Cas9 system followed by the crossing of mice, we generated Svct1 knockout mice lacking both urate transporter 1 and uricase. In the hyperuricemic mice model, serum urate levels were lower than controls, suggesting that Svct1 disruption could reduce serum urate. Given that Svct1 physiologically functions as a renal vitamin C re-absorber, it could also be involved in urate re-uptake from urine, though additional studies are required to obtain deeper insights into the underlying mechanisms. Our findings regarding the dual-substrate specificity of SVCT1 expand the understanding of urate handling systems and functional evolutionary changes in NAT family proteins.

An miR156-regulated nucleobase-ascorbate transporter 2 confers cadmium tolerance via enhanced anti-oxidative capacity in barley

IntroductionCadmium (Cd) is one of the most detrimental heavy metal pollutants, seriously affecting crop production and human health. Nucleobase-ascorbic acid transporters (NAT) are widely present in many living organisms including plants, animals and microbes; however, the role of NAT in plant Cd tolerance remains unknown. ObjectivesTo identify Cd-induced miRNAs that target HvNAT2 and to determine the role of this gene and its product in Cd tolerance. MethodsHigh-throughput-sequencing was used to identify the miRNA expression profile of barley roots in response to Cd stress. Overexpression (OX) and RNAi lines were then constructed for HvNAT2 and comparative transcriptomic analysis was performed to determine the function of this transporter examining its effects on traits such as Cd uptake/flux and translocation, morphology and antioxidant capacity in relation to Cd tolerance. In addition, phylogenetic analysis was performed to obtain insights into the evolution of HvNAT2. ResultsCd stress-induced genome-wide expression profiles of miRNAs identified a Cd-induced miRNA, miR156g-3p_3, that had HvNAT2 as its target. HvNAT2 was negatively regulated in the high-Cd-accumulating and Cd-tolerant genotype Zhenong8. Evolutionary analysis indicated that orthologues of the plasma membrane localized, HvNAT2, can be traced back to the sister group of land plants, the streptophyte algae. Overexpression of HvNAT2 increases Cd tolerance with higher tissue Cd accumulation but less oxidative damage in transgenic barley plants. RNAi of HvNAT2 leads to a significant reduction of Cd tolerance. The higher Cd accumulation in roots of the OX3 line was also demonstrated by confocal microscopy and electrophysiology. Transcriptome analysis showed that the enhancement of antioxidant capacity by HvNAT2 was related to stress signaling pathways. Furthermore, oxidative stress tolerance in HvNAT2-OX plants was regulated by the synthesis of phytochelatins and the glutathione metabolism cycle. ConclusionOur study reveals a key molecular mechanism of NAT in Cd tolerance in plants that is useful for sustainable agricultural production and management of hazardous this heavy metal for better environment management and ecosystem function.

Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein From Rhodococcus erythropolis

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins.
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WITHDRAWN: Foxp3 attenuates cerebral ischemia/reperfusion injury through microRNA-150-5p-modified NCS1

Cerebral ischemia/reperfusion injury (CI/RI) is a pathological process involving complicated molecular mechanisms. We investigated forkhead box P3 (Foxp3)-related mechanism in CI/RI with particular focus on microRNA (miR)-150-5p/nucleobase cation symporter-1 (NCS1) axis. A mouse model was constructed by middle cerebral artery occlusion (MCAO) method. Levels of Foxp3, miR-150-5p and NCS1 were assessed in brain tissues of MCAO mice. By determining the neurological behavior function, neurological deficits, brain tissue pathological characteristics, neuronal apoptosis, inflammatory factors, and oxidative stress-related factors, the functional role of Foxp3, miR-150-5p and NCS1 were evaluated in MCAO mice. The feedback loop was analyzed among Foxp3, miR-150-5p and NCS1. The level of Foxp3 and NCS1 were reduced and that of miR-150-5p was augmented in MCAO mice. Foxp3 bound to miR-150-5p to target NCS1. Up-regulating Foxp3 or NCS1 or suppressing miR-150-5p improved neurological behavior function and neurological deficits, and reduced brain tissue pathological damage, neuronal apoptosis, inflammatory and oxidative stress reactions in MCAO mice. Silencing miR-150-5p or elevating NCS1 decreased Foxp3 silencing-mediated ischemic injury in MCAO mice. Foxp3 is neuroprotective in CI/RI through binding to miR-150-5p to promote NCS1 expression.

The genome of the gymnosperm Picea glauca encodes a single Nucleobase Cation Symporter 1 (PgNCS1) that displays a broad yet unique solute specificity profile

The Picea glauca genome contains a locus that encodes for a nucleobase cation symporter 1 (PgNCS1). As a gymnosperm, P. glauca belongs to a key taxonomic position for an ongoing evolution-function analysis of viridiplantae nucleobase cation symporter 1 proteins (NCS1). Here the solute transport and binding properties for PgNCS1 are determined through heterologous expression in Saccharomyces cerevisiae strains deficient in functional NCS1 loci. PgNCS1 displays a broad, yet unique, solute specificity profile –common with other plant NCS1. Yeast containing PgNCS1 transport adenine, guanine, hypoxanthine, xanthine and uracil and are sensitive to growth on 8-azaadenine. Neither cytosine nor 5 flourocytosine are transported by PgNCS1 but along with caffeine and uric acid, act as competitive inhibitors of [3H]-adenine and [3H]-hypoxanthine uptake. This transporter displays high affinity for adenine (Km = 2.67 μM), guanine (Ki = 1.71 μM) and hypoxanthine (Ki = 1.82 μM) but lesser affinity for xanthine (Ki = 5.36 μM). Arabidopsis plants that are deficient in their endogenous NCS1, yet carry PgNCS1, show significant uptake of [3H]-adenine. The results support previous studies and together confirm a broad nucleobase transport and binding pattern for plant NCS1 across the viridiplantae. PgNCS1 displays a broad yet unique nucleosbase transport and binding profile. It transports adenine, guanine, hypoxanthine, xanthine and uracil but only binds cytosine, caffeine, and uric acid.

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.

Exogenous application of xanthine and uric acid and nucleobase-ascorbate transporter MdNAT7 expression regulate salinity tolerance in apple

BackgroundSoil salinity is a critical threat to global agriculture. In plants, the accumulation of xanthine activates xanthine dehydrogenase (XDH), which catalyses the oxidation/conversion of xanthine to uric acid to remove excess reactive oxygen species (ROS). The nucleobase-ascorbate transporter (NAT) family is also known as the nucleobase-cation symporter (NCS) or AzgA-like family. NAT is known to transport xanthine and uric acid in plants. The expression of MdNAT is influenced by salinity stress in apple.ResultsIn this study, we discovered that exogenous application of xanthine and uric acid enhanced the resistance of apple plants to salinity stress. In addition, MdNAT7 overexpression transgenic apple plants showed enhanced xanthine and uric acid concentrations and improved tolerance to salinity stress compared with nontransgenic plants, while opposite phenotypes were observed for MdNAT7 RNAi plants. These differences were probably due to the enhancement or impairment of ROS scavenging and ion homeostasis abilities.ConclusionOur results demonstrate that xanthine and uric acid have potential uses in salt stress alleviation, and MdNAT7 can be utilized as a candidate gene to engineer resistance to salt stress in plants.

An Erwinia amylovora uracil transporter mutant retains virulence on immature apple and pear fruit

Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. A previous study revealed that an E. amylovora uracil auxotroph was still virulent and can cause disease, suggesting that uracil can be obtained from the host environment. The E. amylovora genome contains a locus encoding for a uracil transporter belonging to the nucleobase cation symporter 2 family, displaying a high level of amino acid sequence similarity to the Escherichia coli UraA. Expression of E. amylovora UraA in nucleobase transporter-deficient E. coli strains, coupled with radiolabeled uptake studies reveal that E. amylovora UraA is a high affinity uracil transporter with a Km of 0.57 μM. Both E. coli and E. amylovora carrying extra copies of E. amylovora UraA are sensitive to growth on the toxic analog 5-fluorouracil. An E. amylovora ΔuraA::Camr mutant is still able to grow and cause disease symptoms on immature pears and apples.

A nucleobase cation symporter 2, EaXanP, from Erwinia amylovora transports xanthine

Erwinia amylovora causes fire blight, one of the more serious diseases for apple and pear cultivation. Previous studies generated E. amylovora mutants in purine metabolic pathway that still retain the ability to grow on host tissue and produce limited disease symptoms. Here we show that the E. amylovora genome has a locus that encodes for a xanthine permease belonging to the nucleobase cation symporter 2 (NCS2) family and the encoded protein displays a high level of amino acid sequence similarity to the Escherichia coli XanP. Our hypothesis is to investigate if the lack of a xanthine transporter has an effect upon disease progression. Heterologous expression of EaXanP in nucleobase transporter-deficient E. coli strains, coupled with radiolabeled nucleobase uptake studies determined that EaXanP is a high affinity xanthine transporter with a Km of 1.2 μM + 0.1 μM that confers sensitivity to growth on caffeine (1,3,7-trimethylxanthine). An E. amylovora ΔxanP::Camr mutant shows resistance to growth on caffeine, while over expression of EaXanP increases growth sensitivity to caffeine. While the EaXanP gene is expressed in infected immature pear fruitlets, an E. amylovora xanthine transport mutant is still able to grow and cause disease symptoms on immature pears and apple fruitlets.

Apple nucleobase cation symporter 1 transports guanine and the toxic guanine analog 6-thioguanine

Erwinia amylovora causes fire blight, a devastating disease of apples and pears. The antimetabolite 6-thioguanine (6 TG) is synthesized and excreted by this bacterium and is involved in pathogenesis. Plants contain numerous nucleobase transporters that readily transport guanine. Studies of the Malus domestica nucleobase cation symporter 1 (MdNCS1) by heterologous expression in nucleobase transport deficient Escherichia coli, reveals transport of adenine, guanine and 6 TG. This is the first report of a plant transporter moving 6-thioguanine, a toxic nucleobase derivative produced by the apple pathogen Erwinia amylovora. MdNCS1 is expressed in both uninfected and E. amylovora infected apple flowers (a common route for pathogen entry) and in E. amylovora infected immature pear fruitlets.

The Nicotiana sylvestris nucleobase cation symporter 1 retains a dicot solute specificity profile

Nucleobase Cation Symporter 1 proteins are present in bacteria, fungi and among plants. Microbes often contain numerous NCS1 proteins that have evolved specific solute specificity profiles (solute transport and binding). Conversely, most members of Viridiplantae have a unique NCS1 that shows a broad, yet species –specific, solute specificity profile. Properties of NCS1 from Nicotiana sylvestris Speg. & Comes (NsNCS1) have been determined as part of an ongoing evolution-function analysis through heterologous expression studies in ncs1-deficient Saccharomyces cerevisiae Meyen ex E.C. Hansen as well as functional assays and subcellular localization studies in planta. NsNCS1 transports the purine adenine with high affinity and moves guanine, uracil, cytosine and hypoxanthine. Xanthine, uric acid and 5-flourocytosine act as competitive inhibitors for NsNCS1. When expressed in Arabidopsis, NsNCS1 locates to the chloroplast and facilitates the transport of guanine and 8-azaguanine. The NsNCS1 solute specificity profile is very similar, yet distinct, from AtNCS1 (A. thaliana (L.) Heynh. NCS1) and indicates that despite evolutionary divergence in the Eudicots - Nicotiana, an Astrid, and Arabidopsis, a Rosid, − little functional difference evolved. The alignment of select plant NCS1 amino acid sequences coupled with a detailed knowledge of each solute specificity profile facilitates identifying residues that may contribute to subtle solute discrimination. Such an evolution-function analysis will complement recent molecular model based site-directed mutagenesis analysis of select microbial and plant NCS1.

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.

Recent developments in nucleobase cation symporter-1 (NCS1) family transport proteins from bacteria, archaea, fungi and plants

The nucleobase cation symporter-1 (NCS1) family of secondary active transport proteins comprises over 2500 sequenced members from bacteria, archaea, fungi and plants. NCS1 proteins use a proton or sodium gradient to drive inward cellular transport of purine and pyrimidine nucleobases and nucleosides, hydantoins and related compounds. The structural organization, substrate binding residues and molecular mechanism of NCS1 proteins are defined by crystal structures of sodium-coupled hydantoin transporter, Mhp1. Plant proteins are most closely related to bacterial/archaeal proteins and the distinct Fur-type and Fcy-type fungal proteins and plant proteins originated through independent horizontal transfers from prokaryotes. Analyses of 25 experimentally characterized proteins reveal high substrate specificity in bacterial proteins, distinct non-overlapping specificities in Fur-type and Fcy-type fungal proteins and broad specificity in plant proteins. Possible structural explanations are identified for differences in substrate specificity between bacterial proteins, whilst specificities of other proteins cannot be predicted by simple sequence comparisons. Specificity appears to be species specific and determined by combinations of effects dictated by multiple residues in the major substrate binding site and gating domains. This is an exploratory research review of evolutionary relationships, function and structural organization, molecular mechanism and origins of substrate specificity in NCS1 proteins and avenues of future direction.

The solute transport and binding profile of a novel nucleobase cation symporter 2 from the honeybee pathogen Paenibacillus larvae.

Here, we report that a novel nucleobase cation symporter 2 encoded in the genome of the honeybee bacterial pathogen Paenibacillus larvae reveals high levels of amino acid sequence similarity to the Escherichia coli and Bacillus subtilis uric acid and xanthine transporters. This transporter is named P. larvae uric acid permease‐like protein (PlUacP). Even though PlUacP displays overall amino acid sequence similarities, has common secondary structures, and shares functional motifs and functionally important amino acids with E. coli xanthine and uric acid transporters, these commonalities are insufficient to assign transport function to PlUacP. The solute transport and binding profile of PlUacP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter‐deficient Saccharomyces cerevisiae strains. PlUacP transports the purines adenine and guanine and the pyrimidine uracil. Hypoxanthine, xanthine, and cytosine are not transported by PlUacP, but, along with uric acid, bind in a competitive manner. PlUacP has strong affinity for adenine K m 7.04 ± 0.18 μm, and as with other bacterial and plant NCS2 proteins, PlUacP function is inhibited by the proton disruptor carbonyl cyanide m‐chlorophenylhydrazone. The solute transport and binding profile identifies PlUacP as a novel nucleobase transporter.

Comparative Genomics, Whole-Genome Re-sequencing and Expression Profile Analysis of Nucleobase:Cation Symporter 2 (NCS2) Genes in Maize.

Nucleobase:cation symporter 2 (NCS2) proteins are important for the transport of free nucleobases, participating in diverse plant growth and developmental processes, as well as response to abiotic stress. To date, a comprehensive analysis of the NCS2 gene family has not been performed in maize. In this study, we conducted a comparative genomics analysis of NCS2 genes in 28 plant species, ranging from aquatic algae to land plants, concentrating mainly on maize. Gene duplication events contributed to the expansion of NCS2 genes from lower aquatic plants to higher angiosperms, and whole-genome/segmental and single-gene duplication events were responsible for the expansion of the maize NCS2 gene family. Phylogenetic construction showed three NCS2 subfamilies, I, II, and III. According to homology-based relationships, members of subfamily I are NCS2/AzgA-like genes, whereas those in subfamilies II and III are NCS2/NATs. Moreover, subfamily I exhibited ancient origins. A motif compositional analysis showed that one symbolic motif (motif 4) of the NCS2/NAT genes was absent in subfamily I. In maize, three NCS2/AzgA-like and 21 NCS2/NAT genes were identified, and purifying selection influenced the duplication of maize NCS2 genes. Additionally, a population genetic analysis of NCS2 genes revealed that ZmNCS2–21 showed the greatest diversity between the 78 inbred and 22 wild surveyed maize populations. An expression profile analysis using transcriptome data and quantitative real-time PCR revealed that NCS2 genes in maize are involved in diverse developmental processes and responses to abiotic stresses, including abscisic acid, salt (NaCl), polyethylene glycol, and low (4°C) and high (42°C) temperatures. ZmNCS2 genes with relatively close relationships had similar expression patterns, strongly indicating functional redundancy. Finally, ZmNCS2–16 and ZmNCS2–23 localize in the plasma membrane, which confirmed their predicted membrane structures. These results provide a foundation for future studies regarding the functions of ZmNCS2 proteins, particularly those with potentially important roles in plant responses to abiotic stresses.

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.