Mapping the transporter-substrate interactions of the Trypanosoma cruzi NB1 nucleobase transporter reveals the basis for its high affinity and selectivity for hypoxanthine and guanine and lack of nucleoside uptake.

Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (Ki) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG0. Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens.

Uniport, Not Proton-Symport, in a Non-Mammalian SLC23 Transporter.

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.

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

BACKGROUND: Soil 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. RESULTS: In 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. CONCLUSION: Our 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.

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.

The guanine-hypoxanthine permease GhxP of Erwinia amylovora facilitates the influx of the toxic guanine derivative 6-thioguanine.

AIM: Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine transporter (EaGhxP) is required for virulence and if it can import the E. amylovora produced toxic analogue 6-thioguanine (6TG) into cells. METHODS AND RESULTS: Characterization of EaGhxP in guanine transport deficient Escherichia coli reveals that it can transport guanine, hypoxanthine and the toxic analogues 8-azaguanine (8AG) and 6TG. Similarly, EaGhxP transports 8AG and 6TG into E. amylovora cells. EaGhxP has a high affinity for 6TG with a Ki of 3·7 µmol l-1 . An E. amylovora ⊿ghxP::Camr strain shows resistance to growth on 8AG and 6TG. Although EaGhxP is expressed during active disease propagation, it is not necessary for virulence as determined on immature apple and pear assays. CONCLUSIONS: EaGhxP is not required for virulence, but it does import 6TG into E. amylovora cells. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of the disease establishment process, E. amylovora synthesizes and exports a toxic guanine derivative 6TG. Our results are counter intuitive and show that EaGhxP, an influx transporter, can move 6TG into cells raising questions regarding the role of 6TG in disease establishment.

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.

Purine Permease-Type Benzylisoquinoline Alkaloid Transporters in Opium Poppy.

Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, benzylisoquinoline alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate alkaloid biosynthesis in opium poppy.

Heterologous expression of the mammalian sodium-nucleobase transporter rSNBT1 in Leishmania tarentolae.

Recombinant expression systems for mammalian membrane transport proteins are often limited by insufficient yields to support structural studies, inadequate post-translational processing and problems related with improper membrane targeting or cytotoxicity. Use of alternative expression systems and optimization of expression/purification protocols are constantly needed. In this work, we explore the applicability of the laboratory strain LEXSY of the ancient eukaryotic microorganism Leishmania tarentolae as a new expression system for mammalian nucleobase permeases of the NAT/NCS2 (Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2) family. We achieved the heterologous expression of the purine-pyrimidine permease rSNBT1 from Rattus norvegicus (tagged at C-terminus with a red fluorescent protein), as confirmed by confocal microscopy and biochemical analysis of the subcellular fractions enriched in membrane proteins. The cDNA of rSNBT1 has been subcloned in a pLEXSY-sat-mrfp1vector and used to generate transgenic L. tarentolae-rsnbt1-mrfp1 strains carrying the pLEXSY-sat-rsnbt1-mrfp1 plasmid either episomally or integrated in the chromosomal DNA. The chimeric transporter rSNBT1-mRFP1 is targeted to the ER and the plasma membrane of the L. tarentolae promastigotes. The transgenic strains are capable of transporting nucleobases that are substrates of rSNBT1 but also of the endogenous L. tarentolae nucleoside/nucleobase transporters. A dipyridamole-resistant Na+-dependent fraction of uptake is attributed to the exogenously expressed rSNBT1.

Uptake of adenine by purine permeases of Coffea canephora.

Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine - but not caffeine - transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium-proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol.

Big Grain3, encoding a purine permease, regulates grain size via modulating cytokinin transport in rice.

Grain size is an important agronomic trait affecting grain yield, but the underlying molecular mechanisms remain to be elucidated. Here, we isolated a dominant mutant, big grain3 (bg3-D), which exhibits a remarkable increase of grain size caused by activation of the PURINE PERMEASE gene, OsPUP4. BG3/OsPUP4 is predominantly expressed in vascular tissues and is specifically suppressed by exogenous cytokinin application. Hormone profiling revealed that the distribution of different cytokinin forms, in roots and shoots of the bg3-D mutant, is altered. Quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that expression of rice cytokinin type-A RESPONSE REGULATOR (OsRR) genes is enhanced in the roots of the bg3-D mutant. These results suggest that OsPUP4 might contribute to the long-distance transport of cytokinin, by reinforcing cytokinin loading into vascular bundle cells. Furthermore, plants overexpressing OsPUP7, the closest homolog of OsPUP4, also exhibited a similar phenotype to the bg3-D mutant. Interestingly, subcellular localization demonstrated that OsPUP4 was localized on the plasma membrane, whereas OsPUP7 was localized to the endoplasmic reticulum. Based on these findings, we propose that OsPUP4 and OsPUP7 function in a linear pathway to direct cytokinin cell-to-cell transport, affecting both its long-distance movement and local allocation.

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.

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.

Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters - the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22 ±â€¯1.7 µM and Vmax of 0.49 ±â€¯0.06 pmol(107 cells)-1 s-1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573 ±â€¯62 nM and Vmax of 0.23 ±â€¯0.06 pmol(107 cells)-1 s-1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family.

NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans.

Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAΔ null mutant exhibits increased sensitivity to these compounds. We provide direct evidence that nmeA overexpression leads to efflux of adenine, xanthine, uric acid and allantoin, the latter two being intermediate metabolites of purine catabolism that are toxic when accumulated cytoplasmically due to relevant genetic lesions. By using a functional GFP-tagged version we show that NmeA is a plasma membrane transporter. Homology modeling and docking approaches identified a single purine binding site and a tentative substrate translocation trajectory in NmeA. Orthologues of NmeA are present in all Aspergilli and other Eurotiomycetes, but are absent from other fungi or non-fungal organisms. NmeA is thus the founding member of a new class of transporters essential for fungal success under specific toxic conditions.

Cryptic purine transporters in Aspergillus nidulans reveal the role of specific residues in the evolution of specificity in the NCS1 family.

NCS1 proteins are H+ or Na+ symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur- and Fcy-like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.

Design and synthesis of purine analogues as highly specific ligands for FcyB, a ubiquitous fungal nucleobase transporter.

In the course of our study on fungal purine transporters, a number of new 3-deazapurine analogues have been rationally designed, based on the interaction of purine substrates with the Aspergillus nidulans FcyB carrier, and synthesized following an effective synthetic procedure. Certain derivatives have been found to specifically inhibit FcyB-mediated [3H]-adenine uptake. Molecular simulations have been performed, suggesting that all active compounds interact with FcyB through the formation of hydrogen bonds with Asn163, while the insertion of hydrophobic fragments at position 9 and N6 of 3-deazaadenine enhanced the inhibition.

Plant development regulated by cytokinin sinks.

Morphogenetic signals control the patterning of multicellular organisms. Cytokinins are mobile signals that are perceived by subsets of plant cells. We found that the responses to cytokinin signaling during Arabidopsis development are constrained by the transporter PURINE PERMEASE 14 (PUP14). In our experiments, the expression of PUP14 was inversely correlated to the cytokinin signaling readout. Loss of PUP14 function allowed ectopic cytokinin signaling accompanied by aberrant morphogenesis in embryos, roots, and the shoot apical meristem. PUP14 protein localized to the plasma membrane and imported bioactive cytokinins, thus depleting apoplastic cytokinin pools and inhibiting perception by plasma membrane-localized cytokinin sensors to create a sink for active ligands. We propose that the spatiotemporal cytokinin sink patterns established by PUP14 determine the cytokinin signaling landscape that shapes the morphogenesis of land plants.

Heterologous complementation studies reveal the solute transport profiles of a two-member nucleobase cation symporter 1 (NCS1) family in Physcomitrella patens.

As part of an evolution-function analysis, two nucleobase cation symporter 1 (NCS1) from the moss Physcomitrella patens (PpNCS1A and PpNCS1B) are examined--the first such analysis of nucleobase transporters from early land plants. The solute specificity profiles for the moss NCS1 were determined through heterologous expression, growth and radiolabeled uptake experiments in NCS1-deficient Saccharomyces cerevisiae. Both PpNCS1A and 1B, share the same profiles as high affinity transporters of adenine and transport uracil, guanine, 8-azaguanine, 8-azaadenine, cytosine, 5-fluorocytosine, hypoxanthine, and xanthine. Despite sharing the same solute specificity profile, PpNCS1A and PpNCS1B move nucleobase compounds with different efficiencies. The broad nucleobase transport profile of PpNCS1A and 1B differs from the recently-characterized Viridiplantae NCS1 in breadth, revealing a flexibility in solute interactions with NCS1 across plant evolution.