Nucleobase transporters (review)

Purines and pyrimidines play a key role in nucleic acid and nucleotide metabolism of all cells. In addition, they can be used as nitrogen sources in plants and many microorganisms. Transport of nucleobases across biological membranes is mediated by specific transmembrane transport proteins. Nucleobase transporters have been identified genetically and/or physiologically in bacteria, fungi, protozoa, algae, plants and mammals. A limited number of bacterial and fungal transporter genes have been cloned and analysed in great detail at the molecular level. Very recently, nucleobase transporters have been identified in plants. In other systems, with less accessible genetics, such as vertebrates and protozoa, no nucleobase transporter genes have been identified, and the transporters have been characterized and classified by physiological and biochemical approaches instead. In this review, it is shown that nucleobase transporters and similar sequences of unknown function present in databases constitute three basic families, which will be designated NAT, PRT and PUP. The first includes members from archea, eubacteria, fungi, plants and metazoa, the second is restricted to prokaryotes and fungi, and the last one is only found in plants. Interestingly, mammalian ascorbate transporters are homologous to NAT sequences. The function of different nucleobase transporters is also described, as is how their expression is regulated and what is currently known about their structure-function relationships. Common features emerging from these studies are expected to prove critical in understanding what governs nucleobase transporter specificity and in selecting proper model microbial systems for cloning and studying plant, protozoan and mammalian nucleobase transporters of agricultural, pharmacological and medical importance.     

Fungal nucleobase transporters

Early genetic and physiological work in bacteria and fungi has suggested the presence of highly specific nucleobase transport systems. Similar transport systems are now known to exist in algae, plants, protozoa and metazoa. Within the last 15 years, a small number of microbial genes encoding nucleobase transporters have been cloned and studied in great detail. The sequences of several other putative proteins submitted to databases are homologous to the microbial nucleobase transporters but their physiological functions remain largely undetermined. In this review, genetic, biochemical and molecular data are described concerning mostly the nucleobase transporters of Aspergillus nidulans and Saccharomyces cerevisiae, the two model ascomycetes from which the great majority of data come from. It is also discussed as to what is known on the nucleobase transporters of the two most significant pathogenic fungi: Candida albicans and Aspergillus fumigatus. Apart from highlighting how a basic process such as nucleobase recognition and transport operates, this review intends to highlight features that might be applicable to antifungal pharmacology.     

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.     

Six related nucleoside/nucleobase transporters from Trypanosoma brucei exhibit distinct biochemical functions

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.     

Using mutants to probe the in vivo function of plastid envelope membrane metabolite transporters

During the last 15 years, much progress has been made in discovering genes encoding solute transporters of the inner plastid envelope membrane. For example, genes encoding transporters for phosphorylated intermediates, dicarboxylates, adenine nucleotides, inorganic anions, and monosaccharides have been cloned. In many cases, the corresponding proteins have been expressed in recombinant host systems for further functional studies, thus allowing detailed in vitro characterization of transporter properties. Knowledge of the gene sequences encoding these transporters have allowed reverse-genetic approaches to study transporter function in vivo. Antisense repression and T-DNA insertion mutagenesis have provided a range of transgenic and mutant plants in which the activity of specific plastid envelope transporters are massively decreased or abolished. Plants with altered transporter activities represent excellent tools to probe the in vivo function of these transporters. Moreover, changing the permeability of the plastid envelope membrane permits the targeted manipulation of subcellular metabolite pools.     

Cloning,heterologous expression,and in situ characterization of the first high affinity nucleobase transporter from a protozoan

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.     

Modelling,substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA

The AzgA purine/H⁺ symporter of Aspergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the Escherichia coli uracil transporter UraA, a member of the nucleobase‐ascorbate transporter (NAT/NCS2) family. The model consists of 14 transmembrane, mostly α‐helical, segments (TMSs) and cytoplasmic N‐ and C‐tails. A distinct compact core of 8 TMSs, made of two intertwined inverted repeats (TMSs 1–4 and 8–11), is topologically distinct from a flexible domain (TMSs 5–7 and 12–14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) might be involved in subsequent substrate translocation, through H⁺ binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA‐like proteins constitute a distant subgroup of the ubiquitous NAT family.     

Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.     

Cloning and functional expression of a gene encoding a P1 type nucleoside transporter from Trypanosoma brucei.

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purines de novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of the TbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites. TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.     

Tonoplast-localized Abc2 transporter mediates phytochelatin accumulation in vacuoles and confers cadmium tolerance

Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates (35)S-PC(2) uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms.     

Expression in an arbuscular mycorrhizal fungus of genes putatively involved in metabolism,transport, the cytoskeleton and the cell cycle

Arbuscular mycorrhizal (AM) fungi are multinucleate, coenocytic, obligate symbionts with no known sexual stages and very wide host and habitat ranges. While contributing vitally to the growth of land plants they face unique challenges in metabolism, transport, growth and development. To provide clues to the strategies that AM fungi have adopted, random sequencing of cDNA's from Glomus intraradices was undertaken. Putative genes for enzymes, transporters, structural proteins and cell-cycle regulatory factors were discovered. Among the EST's of particular interest are sequences with homology to known trehalase, arsenite transporter, cysteine synthase, tubulins, actin, dynein, cell cycle regulatory proteins, and three meiosis-related proteins. The significance of these sequences is discussed in the context of what is known about AM metabolism, transport, growth and phylogeny.     

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.     

Sugar transport across the plasma membranes of higher plants

The fluxes of carbohydrates across the plasma membranes of higher-plant cells are catalysed mainly by monosaccharide and disaccharide-H⁺ symporters. cDNAs encoding these different transporters have been cloned recently and the functions and properties of the encoded proteins have been studied extensively in heterologous expression systems. Several of the proteins have been identified biochemically in these expression systems and their location in plants has been shown immunohistochemically or with transgenic plants which were transformed with reporter genes, expressed under the control of the promoters of individual transporter genes. In this paper we summarize the current knowledge on the molecular biology and biochemistry of higher-plant sugar transport proteins.     

Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.     

Sugar transporters in higher plants--a diversity of roles and complex regulation

Sugar-transport proteins play a crucial role in the cell-to-cell and long-distance distribution of sugars throughout the plant. In the past decade, genes encoding sugar transporters (or carriers) have been identified, functionally expressed in heterologous systems, and studied with respect to their spatial and temporal expression. Higher plants possess two distinct families of sugar carriers: the disaccharide transporters that primarily catalyse sucrose transport and the monosaccharide transporters that mediate the transport of a variable range of monosaccharides. The tissue and cellular expression pattern of the respective genes indicates their specific and sometimes unique physiological tasks. Some play a purely nutritional role and supply sugars to cells for growth and development, whereas others are involved in generating osmotic gradients required to drive mass flow or movement. Intriguingly, some carriers might be involved in signalling. Various levels of control regulate these sugar transporters during plant development and when the normal environment is perturbed. This article focuses on members of the monosaccharide transporter and disaccharide transporter families, providing details about their structure, function and regulation. The tissue and cellular distribution of these sugar transporters suggests that they have interesting physiological roles.     

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.     

Plant ABC transporters

The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators. Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation. Sequences of over 20 plant ABC proteins have been published and include homologues of P-glycoprotein, MRP, PDR5 and organellar transporters. At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised. This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants.     

Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes.

The molecular cloning of cDNAs encoding nucleoside transporter proteins has greatly advanced understanding of how nucleoside permeants are translocated across cell membranes. The nucleoside transporter proteins identified thus far have been categorized into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human members and these will be examined in depth in this review. The human equilibrative nucleoside transporters translocate nucleosides and nucleobases bidirectionally down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. The human concentrative nucleoside transporters cotranslocate nucleosides and sodium unidirectionally against the nucleoside concentration gradients and play a vital role in certain tissues. The regulation of nucleoside and nucleobase transporters is being studied more intensely now that more tools are available. This review provides an overview of recent advances in the molecular biology and regulation of the nucleoside and nucleobase transporters.     

Functional expression of the human hZIP2 zinc transporter

Zinc is an essential nutrient for humans, yet we know little about how this metal ion is taken up by mammalian cells. In this report, we describe the characterization of hZip2, a human zinc transporter identified by its similarity to zinc transporters recently characterized in fungi and plants. hZip2 is a member of the ZIP family of eukaryotic metal ion transporters that includes two other human genes, hZIP1 and hZIP3, and genes in mice and rats. To test whether hZip2 is a zinc transporter, we examined (65)Zn uptake activity in transfected K562 erythroleukemia cells expressing hZip2 from the CMV promoter. hZip2-expressing cells accumulated more zinc than control cells because of an increased initial zinc uptake rate. This activity was time-, temperature-, and concentration-dependent and saturable with an apparent K(m) of 3 microM. hZip2 zinc uptake activity was inhibited by several other transition metals, suggesting that this protein may transport other substrates as well. hZip2 activity was not energy-dependent, nor did it require K(+) or Na(+) gradients. Zinc uptake by hZip2 was stimulated by HCO(3)(-) treatment, suggesting a Zn(2+)-HCO(3)(-) cotransport mechanism. Finally, hZip2 was exclusively localized in the plasma membrane. These results indicate that hZip2 is a zinc transporter, and its identification provides one of the first molecular tools to study zinc uptake in mammalian cells.