Nucleobase Transporter Research Articles

An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes.

Post-transcriptional regulation of gene expression is particularly important in trypanosomatid protozoa. RNA-binding proteins (RBPs) regulate mRNA stability and translation, yet information about how RBPs are able to link environmental cues to post-transcriptional control is scarce. In Trypanosoma brucei, we have previously characterized a short RNA stem-loop cis-element (PuRE, Purine Responsive Element) within the 3′-UTR of the NT8 nucleobase transporter mRNA that is necessary and sufficient to confer a strong repression of gene expression in response to purines. In this study, we have identified a protein complex composed of two RNA-binding proteins (PuREBP1 and PuREBP2) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNA interference results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression.

Current Understanding of the Intestinal Absorption of Nucleobases and Analogs.

It has long been suggested that a Na+-dependent carrier-mediated transport system is involved in the absorption of nucleobases and analogs, including some drugs currently in therapeutic use, for their uptake at the brush border membrane of epithelial cells in the small intestine, mainly based on studies in non-primate experimental animals. The presence of this transport system was indeed proved by the recent identification of sodium-dependent nucleobase transporter 1 (SNBT1/Slc23a4) as its molecular entity in rats. However, this transporter has been found to be genetically deficient in humans and higher primates. Aware of this deficiency, we need to revisit the issue of the absorption of these compounds in the human small intestine so that we can understand the mechanisms and gain information to assure the more rational use and development of drugs analogous to nucleobases. Here, we review the current understanding of the intestinal absorption of nucleobases and analogs. This includes recent knowledge about the efflux transport of those compounds across the basolateral membrane when exiting epithelial cells, following brush border uptake, in order to complete the overall absorption process; the facilitative transporters of equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) may be involved in that in many animal species, including human and rat, without any major species differences.

Purine-responsive expression of the Leishmania donovani NT3 purine nucleobase transporter is mediated by a conserved RNA stem–loop

The ability to modulate gene expression in response to changes in the host environment is essential for survival of the kinetoplastid parasite Leishmania Unlike most eukaryotes, gene expression in kinetoplastids is predominately regulated posttranscriptionally. Consequently, RNA-binding proteins and mRNA-encoded sequence elements serve as primary determinants of gene regulation in these organisms; however, few have defined roles in specific stress response pathways. Leishmania species cannot synthesize purines de novo and must scavenge these essential nutrients from the host. Leishmania have evolved a robust stress response to withstand sustained periods of purine scarcity during their life cycle. The purine nucleobase transporter LdNT3 is among the most substantially up-regulated proteins in purine-starved Leishmania donovani parasites. Here we report that the posttranslational stability of the LdNT3 protein is unchanged in response to purine starvation. Instead, LdNT3 up-regulation is primarily mediated by a 33-nucleotide-long sequence in the LdNT3 mRNA 3' UTR that is predicted to adopt a stem-loop structure. Although this sequence is highly conserved within the mRNAs of orthologous transporters in multiple kinetoplastid species, putative stem-loops from L. donovani and Trypanosoma brucei nucleobase transporter mRNAs were not functionally interchangeable for purine-responsive regulation. Through mutational analysis of the element, we demonstrate that species specificity is attributable to just three variant bases within the predicted loop. Finally, we provide evidence that the abundance of the trans-acting factor that binds the LdNT3 stem-loop in vivo is substantially higher than required for regulation of LdNT3 alone, implying a potential role in regulating other purine-responsive genes.

Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells

Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective nucleobase transporter. Although it is highly expressed in the liver, its role in nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 μM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 μM, which was comparable to that of 2.30 μM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.

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.

Specificity profile of NAT/NCS2 purine transporters in Sinorhizobium (Ensifer) meliloti.

Sinorhizobium (Ensifer) meliloti is a model example of a soil alpha-proteobacterium which induces the formation of nitrogen-fixing symbiotic nodules on the legume roots. In contrast to all other rhizobacterial species, S. meliloti contains multiple homologs of nucleobase transporter genes that belong to NAT/NCS2 family (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2). We analyzed functionally all (six) relevant homologs of S. meliloti 1,021 using Escherichia coli K-12 as a host and found that five of them are high-affinity transporters for xanthine (SmLL9), uric acid (SmLL8, SmLL9, SmX28), adenine (SmVC3, SmYE1), guanine (SmVC3), or hypoxanthine (SmVC3). Detailed analysis of substrate profiles showed that two of these transporters display enlarged specificity (SmLL9, SmVC3). SmLL9 is closely related in sequence with the xanthine-specific XanQ of E. coli. We subjected SmLL9 to rationally designed site-directed mutagenesis and found that the role of key binding-site residues of XanQ is conserved in SmLL9, whereas a single amino-acid change (S93N) converts the xanthine/uric-acid transporter SmLL9 to a xanthine-preferring variant, due to disruption of an essential hydrogen bond with the C8 oxygen of uric acid. The results highlight the presence of several different purine nucleobase transporters in S. meliloti and imply that the purine transport might be important in the nodule symbiosis involving S. meliloti.

SLC43A3 Is a Biomarker of Sensitivity to the Telomeric DNA Damage Mediator 6-Thio-2'-Deoxyguanosine.

Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2'-deoxyguanosine (6-thio-dG). A large panel of non-small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (SLC43A3), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of SLC43A3 mRNA predicted sensitivity to 6-thio-dG and therefore SLC43A3 could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. SIGNIFICANCE: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2'-deoxyguanosine.

Updates on human equilibrative nucleoside transporter 1 in neoplastic diseases

The human equilibrative nucleoside transporter 1(hENT1)is a multi-integrated membrane protein that mediates the transport of nucleosides, nucleobases and nucleoside analogs.It is mainly distributed on the cell membrane in humans.hENT1 maintains the homeostasis of cells and the intracellular concentration of nucleoside drugs through facilitating diffusion, thereby affecting DNA and RNA synthesis, cell signaling, metabolic regulation and tumor chemotherapy.This article reviews the progress in the structure and function of hENT1, the distribution in tumor tissues, the nucleoside analogs response and the prognosis of neoplastic diseases. Key words: Human equilibrative nucleoside transporter 1; Nucleoside analogs; Neoplasm

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter.

6-Mercaptopurine (6-MP) is a nucleobase analog used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disorders. However, the mechanisms underlying its transport into target cells have remained elusive. The protein encoded by SLC43A3_1 [equilibrative nucleobase transporter 1 (ENBT1)] has recently been shown to transport endogenous nucleobases. A splice variant (SLC43A3_2), encoding a protein with 13 additional amino acids in the first extracellular loop, is also expressed but its function is unknown. We hypothesized that 6-MP is a substrate for both variants of ENBT1. Human embryonic kidney 293 (HEK293) cells (lacking endogenous ENBT1 activity) were transfected with each of the coding region variants of SLC43A3. ENBT1 function was assessed via the rate of flux of [3H]adenine and [14C]6-MP across the plasma membrane. Both SLC43A3 variants encoded proteins with similar functional properties. [14C]6-MP and [3H]adenine had K m values (±S.D.) of 163 ± 126 and 37 ± 26 µM, respectively, for this system. Decynium-22, 6-thioguanine, and 6-methylmercaptopurine inhibited 6-MP uptake with K i values of 1.0 ± 0.4, 67 ± 30, and 73 ± 20 µM, respectively. ENBT1 also mediated adenine-sensitive efflux of 6-MP from the SLC43A3-HEK293 cells. MRP4 also contributed to the efflux of 6-MP in this model, but was less efficient than ENBT1 in this regard. Furthermore, transfection of HEK293 cells with SLC43A3 increased the sensitivity of the cells to the cytotoxic effects of 6-MP by more than 7-fold. Thus, both variants of ENBT1 are key players in the transfer of 6-MP into and out of cells, and changes in SLC43A3 expression impact 6-MP cytotoxicity.

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.

NAT/NCS2-hound: a webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase-cation symporters of the NAT/NCS2 family.

Nucleobase transporters are important for supplying the cell with purines and/or pyrimidines, for controlling the intracellular pool of nucleotides, and for obtaining exogenous nitrogen/carbon sources for metabolism. Nucleobase transporters are also evaluated as potential targets for antimicrobial therapies, since several pathogenic microorganisms rely on purine/pyrimidine salvage from their hosts. The majority of known nucleobase transporters belong to the evolutionarily conserved and ubiquitous nucleobase-ascorbate transporter/nucleobase-cation symporter-2 (NAT/NCS2) protein family. Based on a large-scale phylogenetic analysis that we performed on thousands of prokaryotic proteomes, we developed a webserver that can detect and distinguish this family of transporters from other homologous families that recognize different substrates. We can further categorize these transporters to certain evolutionary groups with distinct substrate preferences. The webserver scans whole proteomes and graphically displays which proteins are identified as NAT/NCS2, to which evolutionary groups and subgroups they belong to, and which conserved motifs they have. For key subgroups and motifs, the server displays annotated information from published crystal-structures and mutational studies pointing to key functional amino acids that may help experts assess the transport capability of the target sequences. The server is 100% accurate in detecting NAT/NCS2 family members. We also used the server to analyze 9,109 prokaryotic proteomes and identified Clostridia, Bacilli, β- and γ-Proteobacteria, Actinobacteria, and Fusobacteria as the taxa with the largest number of NAT/NCS2 transporters per proteome. An analysis of 120 representative eukaryotic proteomes also demonstrates the server's capability of correctly analyzing this major lineage, with plants emerging as the group with the highest number of NAT/NCS2 members per proteome.

Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche

BackgroundTrypanosoma cruzi, the causative agent of Chagas' disease is unable to synthesise its own purines and relies on salvage from the host. In other protozoa, purine uptake has been shown to be mediated by Equilibrative Nucleoside Transporters (ENTs). MethodsTo investigate the functionality of T. cruzi-encoded ENT transporters, its four putative ENT genes (TcrNB1, TcrNB2, TcrNT1 and TcrNT2) were cloned and expressed in genetically adapted Trypanosoma brucei procyclic cells from which the nucleobase transporter locus was deleted. ResultsTcrNB1 displayed very high affinity for hypoxanthine (Km 93.8 ± 4.7 nM for) and guanine, and moderate affinity for adenine. TcrNT1 was found to be a high-affinity guanosine/inosine transporter (inosine Km is 1.0 ± 0.03 μM; guanosine Ki is 0.92 ± 0.2 μM). TcrNT2 encoded a high-affinity thymidine transporter (Km = 223.5 ± 7.1 nM) with a clear preference for 2’-deoxypyrimidines. TcrNB2, whose activity could not be determined in our system, could be a low-affinity purine nucleobase transporter, given its sequence and predicted structural similarities to Leishmania major NT4. All 4 transporter genes were highly expressed in the amastigote forms, with much lower expression in the non-dividing stages. ConclusionsThe data appear to show that, surprisingly, T. cruzi has a preference for oxopurines over aminopurines and efficiently transports 2′-deoxypyrimidines. The T. cruzi ENTs display exceptionally high substrate affinity as an adaptation to their intracellular localisation. General significanceThis study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter.

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.

Intestinal Nucleoside Transporters: Function, Expression, and Regulation.

The gastrointestinal tract is the absorptive organ for nutrients found in foods after digestion. Nucleosides and, to a lesser extent nucleobases, are the late products of nucleoprotein digestion. These metabolites are absorbed by nucleoside (and nucleobase) transporter (NT) proteins. NTs are differentially distributed along the gastrointestinal tract showing also polarized expression in epithelial cells. Concentrative nucleoside transporters (CNTs) are mainly located at the apical side of enterocytes, whereas equilibrative nucleoside transporters (ENTs) facilitate the basolateral efflux of nucleosides and nucleobases to the bloodstream. Moreover, selected nucleotides and the bioactive nucleoside adenosine act directly on intestinal cells modulating purinergic signaling. NT-polarized insertion is tightly regulated. However, not much is known about the modulation of intestinal NT function in humans, probably due to the lack of appropriate cell models retaining CNT functional expression. Thus, the possibility of nutritional regulation of intestinal NTs has been addressed using animal models. Besides the nutrition-related role of NT proteins, orally administered drugs also need to cross the intestinal barrier, this event being a major determinant of drug bioavailability. In this regard, NT proteins might also play a role in pharmacology, thereby allowing the absorption of nucleoside- and nucleobase-derived drugs. The relative broad selectivity of these membrane transporters also suggests clinically relevant drug-drug interactions when using combined therapies. This review focuses on all these physiological and pharmacological aspects of NT protein biology. © 2017 American Physiological Society. Compr Physiol 8:1003-1017, 2018.

Structure-activity relationships in fungal nucleobases transporters as dissected by the inhibitory effects of novel purine analogues

We have previously rationally designed, synthesized and tested a number of 3-deazapurine analogues, which inhibit the ubiquitous fungal nucleobase transporter FcyB, through binding in its major substrate binding site, by specifically interacting with Asn163. Here, in an effort to further understand the molecular details of structure-activity relationships in all three major nucleobase transporters of fungi, we extend this study by designing, based on our previous experience, synthesizing and testing further 3-deazapurine analogues. We thus identify seven new compounds with relatively high affinity (19–106 μΜ) for the FcyB binding site. Importantly, four of these compounds can also efficiently inhibit AzgA, a structurally and evolutionary distinct, but functionally similar, purine transporter. Contrastingly, none of the new compounds tested had any effect on the transport activity of the uric acid-xanthine transporter UapA, albeit this being a structural homologue of AzgA. Besides the apparent importance for understanding how nucleobase transporter specificity is determined at the molecular level, our work might constitute a critical step in the design of novel purine-related antifungals.

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.

Urate transport function of rat sodium-dependent nucleobase transporter 1.

Sodium‐dependent nucleobase transporter 1 (SNBT1) is a nucleobase‐specific transporter identified in our recent study. In an attempt to search for its potential substrates other than nucleobases in this study, we could successfully find urate, a metabolic derivative of purine nucleobases, as a novel substrate, as indicated by its specific Na+‐dependent and saturable transport, with a Michaelis constant of 433 μmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin‐Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co‐introduction of rat breast cancer resistance protein (rBCRP), a urate efflux transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.

The PLUTO plastidial nucleobase transporter also transports the thiamin precursor hydroxymethylpyrimidine

In plants, the hydroxymethylpyrimidine (HMP) and thiazole precursors of thiamin are synthesized and coupled together to form thiamin in plastids. Mutants unable to form HMP can be rescued by exogenous HMP, implying the presence of HMP transporters in the plasma membrane and plastids. Analysis of bacterial genomes revealed a transporter gene that is chromosomally clustered with thiamin biosynthesis and salvage genes. Its closest Arabidopsis homolog, the plastidic nucleobase transporter (PLUTO), is co-expressed with several thiamin biosynthetic enzymes. Heterologous expression of PLUTO in Escherichia coli or Saccharomyces cerevisiae increased sensitivity to a toxic HMP analog, and disrupting PLUTO in an HMP-requiring Arabidopsis line reduced root growth at low HMP concentrations. These data implicate PLUTO in plastidial transport and salvage of HMP.