Molecular Evolution of the Equilibrative Nucleoside Transporter Family: Identification of Novel Family Members in Prokaryotes and Eukaryotes

Yugo Acimovic* and Imogen R. Coe{dagger},2

*The Centre for Computational Biology, Hospital for Sick Children, Toronto;
{dagger}Department of Biology, York University, Toronto


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Acknowledgements
 References
 
Equilibrative nucleoside transporters (ENTs) are integral membrane proteins which enable the movement of hydrophilic nucleosides and nucleoside analogs down their concentration gradients across cell membranes. ENTs were only recently characterized at the molecular level, and little is known about the tertiary structure or distribution of these proteins in nonmammalian organisms. To identify conserved regions, residues, and motifs of ENTs that may indicate functionally important parts of the protein and to better understand the evolutionary history of this protein family, we conducted an exhaustive analysis to characterize and compare ENTs in taxonomically diverse organisms. We have identified novel ENT family members in humans, mice, fish, tunicates, slime molds, and bacteria. This greatly extends our knowledge on the distribution of the ENTs in eukaryotes, and we have identified, for the first time, family members in bacteria. The prokaryote ENTs are attractive models for future studies on transporter tertiary structure and mechanism of substrate translocation. Using sequence similarities, we have identified regions, residues, and motifs that are conserved across all family members. These areas are presumably correlated with function and therefore are important targets for future analysis. Finally, we propose an evolutionary history for the ENT family which clarifies the origin(s) of multiple isoforms in different taxa.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Acknowledgements
 References
 
Transmembrane transport is a critically important physiological process in all cells and is likely to have evolved early to allow for controlled uptake and release of nonlipophilic compounds. Membrane transporters are responsible for the uptake of essential nutrients, modulation of concentrations of physiologically relevant chemicals, and active release of substances such as signaling molecules. Equilibrative nucleoside transporters (ENTs) are a unique family of proteins with no apparent sequence homology to other types of transporters, which enable facilitated diffusion of nucleosides, such as adenosine, and nucleoside analogs used in chemotherapy across cell membranes (Hyde et al. 2001Citation ). Some mammalian ENTs have been well characterized at the molecular and pharmacological levels (Griffiths et al. 1997Citation ; Crawford et al. 1998Citation ), and a number of isoforms (ENT1–3) are known to exist (Hyde et al. 2001Citation ). In humans, ENT isoforms show differential tissue distribution (Pennycooke et al. 2001Citation ) and differing substrate specificity (Hyde et al. 2001Citation ). Because they are likely to be responsible for the uptake of the majority of nucleoside analog drugs used clinically, an increased understanding of the physiological significance and the role played by multiple family members within the same species and even the same tissue is needed. The development of nonhuman and nonmammalian model systems may facilitate studies on the physiological relevance of different ENT isoforms.

ENT-like proteins have been identified in nonmammalian organisms, although most of these homologs remain functionally uncharacterized. Whether these ENTs are paralogs (homologs that have arisen by gene duplication) or orthologs (homologs which have arisen by speciation) is not clear. Moreover, the origin of the ENTs is elusive because they have not been identified in prokaryotes. Despite the increasing number of ENT homologs that have been identified in an increasing diversity of organisms, a comprehensive analysis of the evolution of this protein family has not yet been conducted.

Evolutionary studies of protein families can be instrumental in determining regions of primary structural conservation, suggesting parts of the protein which are functionally important. Additionally, an increased understanding of the evolutionary history of this protein family may indicate the presence of novel, undiscovered family members in mammals, and such studies may help elucidate the physiology of novel forms because orthologs are often present in similar subcellular locations (Marcotte et al. 2000Citation ).

Therefore, we have undertaken a comprehensive analysis of publicly accessible databases to identify as many members of the ENT family as possible and to identify evolutionarily conserved regions which are suggestive of functional importance. Based on the relatedness of these sequences, we propose an evolutionary history for the ENT family which defines the possible origins of the ENT isoforms present in mammals.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Acknowledgements
 References
 
Generation of Database
Sequences were collected by exhaustive, word-based ENTREZ searches in GenBank, using any combination of words related to nucleoside transporters, e.g., nucleoside, purine, pyrimidine, cytidine, adenosine, guanosine, transport, transporter, etc. The compiled sequences were made nonredundant by unique sort (Shell script). Blast searches were then performed using selected sequences as the query to pick out highly related and relevant sequences which would not have been recognized by the search described above.

Other sequence databases that are publicly accessible were also subjected to a similar database mining approach. These databases include those operated by the Joint Genome Institute, (http://www.jgi.doe.gov/) which is responsible for the Fugu Genome Project and Ciona Genome Project. Mycetozoan sequences were obtained from the Dictystelium discoideum cDNA Project (http://www.csm.biol.tsukuba.ca.jp). Available nonhuman sequence databases at The Institute for Genome Research (TIGR, www.tigr.org) were mined in a similar manner. Databases were subjected to a reverse screening approach in which putative ENTs from evolutionarily distant taxa were used to rescreen for novel ENTs in mammals (i.e., lower eukaryotic ENTs used as query sequences in Blast searches of GenBank). Protein sequences that were of full length or apparently of full length (presence of initial methionine) were included preferentially over partial sequences, although the latter were included for some organisms. All sequences were loaded into a custom-built database (nDb) in C language in FASTA or GenBank (gb) format. The nDb and supporting procedures were implemented mainly in C programming language with some code written in Perl. GenBank flat files were used because they contain more information about molecules (i.e., short name, organism name, mapping information, etc.), can be parsed out by program, need not be assigned manually, and therefore are more suitable for automatic uploads. The nDb was used to keep sequences organized, allowing for easy update, removal, or insertion of sequences, and also to produce desired, appropriately formatted output.

Naming of Sequences
Of the proteins that have been identified based on sequence similarity, only a few have been functionally characterized (with designated names, hENT1, CeENT1, etc.). In this study we describe all family members (determined based on sequence homology) as NTs and define them, according to the conventions of this field (e.g., Hyde et al. 2001Citation ), using an abbreviation of genus and species name followed by the designation ENT (e.g., Drosophila melanogaster, DmENT1). Numbering of sequences (e.g., ENT1, ENT2, etc.) does not imply homology (i.e., hENT1 and DmENT1 are not necessarily orthologs) but rather chronology of discovery or identification. For clarity, we have not distinguished in the naming of ENTs between those which have been functionally characterized and those which have not. Rather, we suggest that all proteins described in this study, which have not been functionally characterized, be considered as putative ENTs, until confirmed as nucleoside transporters by appropriate functional assays.

Multiple Alignments and Phylogentic Analysis
For the alignment of sequences from organisms that are more closely related (in evolutionary terms), such as all mammalian ENTs, all vertebrate ENTs, etc., we used CLUSTAL W, with default settings for gap opening (10.0) and gap extension (0.1–0.2) penalties in both pairwise and multiple alignments using the Blosum Protein Matrix. Alignments were visualized in CLUSTAL X. After the alignment, we used the Neighbor Joining (N-J) method of Saitou and Nei (1987)Citation to generate trees in CLUSTAL X. Data were bootstrapped (random # 111, at 1,000 repeats), and the resulting tree was visualized using Phylip drawgram and drawtree (Felsenstein 1989Citation ). In preliminary analyses, Phylip was also used, along with Dialign (Morgenstern 1999Citation ), to determine if phylogenies generated by these programs were consistent with CLUSTAL W. We found that all three programs produced comparable, equivalent trees and therefore continued our analysis with CLUSTAL as our tool of choice.

For the analysis of sequences from evolutionarily distant organisms (e.g., prokaryotes and mammals) or the entire database, we used CLUSTAL W with gap opening penalties of 4.0 and 5.0 and gap extension penalties of 0.1 and 0.2 for pairwise and multiple alignments, respectively. These gap opening and extension parameters, as well as our choice of matrices (Blosum) were selected as optimal by running the Smith-Waterman algorithm on every pair in our set and then observing the overall best identity and similarity scores. A set of PHAT matrices (Ng, Henikoff, and Henikoff 2000Citation ), specific for transmembrane proteins, yielded results that were similar to those of Blosum. Alignments are presented as CLUSTAL X outputs. Protein comparisons (% identity or similarity, or both) were determined by using the local alignment analysis program, water (found at EMBOSS, http://www.uk.embnet.org/Software/EMBOSS/), which uses the Smith-Waterman algorithms for finding optimal alignments. Large-scale analyses were run on our supercomputer at MENDEL.ocgc.ca.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Acknowledgements
 References
 
Generation of Database
A database consisting of sequences considered to be members of the ENT protein family (as of May 14, 2002) was compiled (table 1 Go ). Our database consists of sequences derived from representatives of eukaryotes plus four prokaryotic sequences. We compiled a total of more than 45 different sequences (table 1 ) which consisted of 17 functionally characterized ENTs and 29 other proteins which were included in our analyses based on their sequence similarity. Splice variants and partial sequences for the mammalian ENTs are included in the table but are not numbered as additional, unique ENTs (e.g., table 1 , #2i HNP36, #2ii hENT2v). It is currently not clear whether these putative proteins exist as functional ENTs.


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Table 1 The ENT Family

 

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Table 1 Continued

 
Novel Mammalian ENTs
We identified two novel, previously unknown, mammalian ENTs, which we have named hENT4 (table 1 , #4) and mENT4 (table 1 , #10), using a reverse screening approach, whereby the partial Dictyostelium sequence (table 1 , #41) was compared with the entire GenBank database. Our initial aim in doing this was to identify a full-length mycetozoan sequence. However, remarkably, among the sequences with highest homology to the Dictyostelium fragment, DdENT, were the mouse protein (table 1 , #10, gi:19343703) and the Drosophila homolog, DmENT3 (table 1 , #16). The mouse protein is annotated as a putative nucleoside transporter and has the prototypic 11 transmembrane (TM) domain structure when analyzed using TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), a web-based server for the prediction of transmembrane helices in proteins (Krogh et al. 2001Citation ). Therefore, we concluded that this mouse protein is a member of the ENT family. We then used the mENT4 to identify the apparent human ortholog which has only recently been deposited in GenBank (table 1 , #4, 4i, 4ii). At this time (May 14, 2002), the hENT4 genomic, mRNA, and predicted protein sequence data appear to be incomplete. A small portion of the N-terminal of hENT4 (correlating approximately to amino acids 56–121) shows very high sequence identity with phosphorylase kinase, whereas the rest of hENT4 is clearly related to the other members of the ENT family. Because mENT4 shows no similarity with this phosphorylase kinase, this feature of hENT4 may be an artifact of cloning or may be due to some unusual genetic event. Characterization of the hENT4 gene would help elucidate it's origin and phylogenetic history, but at this time the structure and sequence of the gene, located on chromosome 7, are unclear because the contigs in this area appear to be out of order. We have not identified ENT4 in other mammals but are continuing to monitor the database with respect to hENT4 and its homologs.

The ENT4 proteins are similar to the other mammalian ENTs (fig. 1 ) but have overall low homology to ENT1/2/3. For example, the mouse sequence, mENT4, has 28% identity and 41% similarity with the mammalian ENTs, except for hENT4 for which there is 54.9% identity and 60.9% similarity overall. Similar levels of identity-similarity exist for hENT4, relative to the other mammalian ENT, although this analysis is compromised by the unusual features of the protein noted above. The mouse ortholog is longer (528 amino acids) than any of the other mENTs (456–475 amino acids) due to extended amino and carboxy temini as well as a longer putative cytoplasmic loop between the predicted TM domains 6 and 7. Despite the low overall similarity at the primary level, the predicted topology of the ENT4s correlates with the prototypic 11 TM structure defined for mammalian and invertebrate ENTs (Sankar et al. 2002Citation ). In addition, mENT4 shows high homology with the fish ortholog, FrENT4 (46.9% identity, 56.8% similarity), compared with the other fish sequences (approximately 25% identity, 38% similarity) and, most remarkably, a higher degree of similarity with the Drosophila ortholog, DmENT3 (33.7% identity, 45.9% similarity), than with other mENTs. This suggests that the mammalian ENT4 lineage is evolutionarily distinct and ancient compared with the ENT1/2/3 lineage(s).



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Fig. 1.—Alignment of the human and mouse ENTs (table 1 , #1–4 and #7–10). Regions 1–4 were the regions of highest conservation across all sequences in the database and were used in subsequent analyses. Lines and numbers under sequences indicate putative transmembrane domains. Annotations above the sequence are according to Clustal X (1.8) output. Asterisks (*) indicate positions which have a single, fully conserved residue. Colons (:) indicate conservation of strong groups, periods (.) indicate conservation of weak groups

 
Novel Fish ENTs
We have identified the first nonmammalian, vertebrate members of the ENT family in the pufferfish, Fugu rubripes, from sequences that are available as a part of its genome sequencing project (table 1 , #12–12iv, FrENT1–FrENTx). Incomplete and partial sequences of the putative ENTs of pufferfish are not numbered separately because we cannot confirm that these are the complete, accurate, and independent sequences at this time (May 14, 2002). However, the longer sequences (which, although incomplete, are more comparable with mammalian ENTs, FrENT1–4) were included in analyses, where possible, to help elucidate phylogenies for the different isoforms. We searched for additional fish putative ENTs in the Danio rerio (zebrafish) database (http://www.sanger.ac.uk/Projects/D_rerio/) but were unable to find zebrafish ENT orthologs. This is most likely to be due to the incomplete state of this database. The four fish ENTs (FrENT1–4) were compared with the mammalian ENTs and found to cluster into family or isoform-specific groups (fig. 2 ) based on similarity (i.e., FrENT3 clusters with hENT3, etc.). However, in this analysis, FrENT1 and FrENT2 appear to be equally similar to the ENT1 and ENT2 clusters. This suggests that either FrENT1 and FrENT2 are paralogs, possibly as a consequence of a genome duplication in the teleost ancestor (Taylor et al. 2001)Citation , or that the ENT1/2 split occurred after the appearance of the teleosts, such that FrENT1 and FrENT2 are orthologs of the mammalian ENT1/2 precursor. Because our fish sequences are not complete and ENTs from additional polyploid or tetraploid organisms such as zebrafish or Xenopus are not yet available, this part of the evolutionary history of the ENT family remains unresolved.



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Fig. 2.—Rootless phylogeny of vertebrate ENTs (based on #1–12, table 1 ) showing clustering of ENT isoform families

 
Novel Tunicate ENTs
Several partial ENT-like sequences were identified in the tunicate, Ciona intestinalis (table 1 , #13, CiENT). Although these sequences are incomplete, we numbered them as independent and separate sequences and included them in the protein analysis (e.g., fig. 3a ) due to the significance of the urochordata as representatives of the most primitive chordate phylum and the absence of previous reports of ENT homologs from this group. Whether the ENT-like fragments come from independent proteins or represent parts of the same protein remains to be determined. However, our data clearly demonstrate the existence of well-conserved ENTs in this taxon.



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Fig. 3.—Alignments of ENTs from representatives of mammals, fish, invertebrates, plants, and prokaryotes. (a) Region 1—alignment of 39 ENTs; (b)) Region 2—alignment of 31 ENTs plus E. coli Tsx; (c) Region 3—alignment of 42 ENTs plus E. coli and Enterobacter aerogenes Tsx.Alignments highlight conserved regions in sequences that span these regions without redundancy (i.e., only hENT1, not hENT2, is shown in panel b because these two sequences are almost identical in this region). Asterisks indicate residues that are identical in Tsx and at least one other ENT family member; arrows indicate highly conserved residues in all or most ENTs, ''' indicates hydrophobic regions in all or most ENTs

 
Novel Mycetozoan ENTs
The slime mold (Dicyostelium discoideum) ENT homolog, DdENT (table 1 , #41), is also an incomplete sequence but was numbered independently and included in the analysis because ENT homologs from this organism have not previously been reported and therefore are important in understanding the evolution of the ENT family. The Dictystelium (DdENT) fragment extends across regions 1 and 2 (fig. 3a and b ) and is clearly homologous to other ENTs.

Novel Prokaryote ENTs
Putative prokaryotic ENT family members were initially identified based on their functional characterization as nucleoside-specific channels (Bremer et al. 1990Citation ; Nieweg and Bremer 1997Citation ). The Tsx proteins are found in the outer membrane of gram-negative bacteria, such as Escherichia coli (Ec Tsx) and Enterobacter aerogenes (Ea Tsx), where they function as energy-independent, substrate-specific transporters for nucleosides and deoxynucleosides (Bremer et al. 1990Citation ; Nieweg and Bremer 1997Citation ). Despite their designation as nucleoside transport proteins, analysis of similarity between Tsx proteins and other ENTs, using default setting for BLAST or CLUSTAL W, showed no significant homology across the length of the entire protein. However, by removing the signal sequence and adjusting the analysis parameters, regions of similarity between Ec Tsx (table 1 , #42) and Ea Tsx (table 1 , #43) to the rest of the ENT family become apparent, particularly in regions 2 and 3 (fig. 3b and c ). In region 2 (fig. 3b ), 11 of 27 amino acids in Ec Tsx are identical to an amino acid in at least one (and usually more than one) other ENT family member, and three glycine residues are conserved in all ENTs from prokaryotes to humans. In region 3 (fig. 3c ), 18 of 29 amino acids are identical in the prokaryotes to at least one other ENT family member. A tryptophan (W, +3), a hydrophobic stretch in front of an asparagine (N, +14), an aspartate (D, +16), and a glycine (G, +18) are highly conserved across the 42 ENTs included in this analysis. In general, the Tsx proteins showed greatest similarity with the ENTS identified in the alveolate, apicomplexan protists, Plasmodium falciparum (table 1 , #30, PfENT1) and Toxoplasma gondii (table 1 , #31, TgAT), which are themselves somewhat distinct in sequence from the rest of the ENT family, reflecting the unique nature of the selective pressures on protozoan parasites. We could not identify, with confidence, ENT homologs on the basis of sequence similarity in any other bacteria or among the archaea.

Following our determination that the Tsx proteins could be prokaryotic members of the ENT family, we investigated their putative structure to determine if their predicted topology supported this designation. Because these are bacterial outer membrane proteins, standard analysis programs for determining TM domains (such as TMHMM 2.0) are not appropriate. Therefore, we used a program for predicting membrane protein topology based on crystal structure of other outer membrane proteins such as FhuA and FepA (Diederichs et al. 1998Citation , http://strucbio.biologie.uni-konstanz.de/~kay/om_topo_predict2.html). Based on this analysis, Ec Tsx is predicted to have 14 TM domains, with both N- and C-terminal regions located within the periplasm (fig. 4 ). A large periplasmic loop exists between putative TM domains 6 and 7, and the conserved glycines found in region 2 are located within the loop, within TM domain 8, and between TM 7 and 8. Overall, this topology is reminiscent of the prototypic eukaryotic ENT structure (Sankar et al. 2002Citation ).



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Fig. 4.—Predicted topology of E. coli Tsx. Grey blocks indicate putative transmembrane domains. Conserved glycines in all ENTs in region 2 shown by asterisks

 
Additional Novel ENTs
In addition to ENT members numbered independently in table 1 , a number of tentative ENT orthologs from additional taxa were identified in the TIGR Orthologous Gene Alignment (TOGA) database (http://www.tigr.org/tdb/toga). We have noted these in table 1 but have not included them in further analyses because they are short, incomplete, unannotated sequences based on EST data. However, we recommend this site as a source of putative ENTs, from a diverse array of taxa, which will be amenable for future analyses.

Phylogenetic Analysis and Homology of ENTs
We have compiled a database of ENTs, based on sequence similarity, which includes representatives from vertebrate, invertebrate, and fungal eukaryotes plus four prokaryote sequences. Phylogenetic analysis of these ENTs, some partial and possibly inaccurate, from such a diverse assortment of organisms, with variable or, in many cases, very low levels of sequence similarity, is problematic. To develop a model for the possible evolutionary history of this family, we used several approaches including analyses (and derived phylogenies) based of subsets of the data (e.g., all mammals, all vertebrates, etc.) combined with analyses (and derived phylogenies) based on regions of highest conservation in all sequences. Analysis and alignment of all the ENTs in the database resulted in the identification of four regions (regions 1–4) of high conservation (shown for the mouse and human ENTs in fig. 1 ) which were used in subsequent analyses of either the entire database or the subsets thereof (figs. 3a, 3b, 3c, and 5a ). A number of conserved residues and patterns were identified in these regions in the majority of the ENTs in our database (fig. 3a, 3b, 3c, and 5a ). Region 4 (fig. 5a ) was the most extensive region (in terms of length), with a high degree of homology for a broad range of taxa. Region 4 was used to generate a phylogeny for the ENT family (fig. 5b ) which clearly shows the distinction between the ENT1/2/3 lineages and the ENT4 lineage, and the many taxon-specific duplications that have taken place to generate paralogs within species. Based on these analyses in combination with other phylogenies derived from regions 1, 2, and 3 (data not shown), we propose a model of the evolutionary history of the ENT family from prokaryotes to mammals (fig. 6 ).



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Fig. 5.—(a) Region 4—alignment of 41 ENTs; symbols as described for figure 3 ; (b) rootless phylogeny derived from Region 4.

 


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Fig. 6.—Putative evolutionary history of the ENT family. Asterisks indicate putative taxon-specific gene duplication events leading to ENT paralogs; questions marks indicate putative gene or genome duplication events leading to new ENT lineage. No time frame is implied by lengths of branches

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 Acknowledgements
 References
 
The first ENT was identified at the molecular level 5 years ago (Griffiths et al. 1997Citation ). Since that time, the explosion in bioinformatics has provided an abundance of sequence data for both nucleic acids and proteins in a diverse array of organisms. Within these data is a wealth of information relating to ENT structure, and possibly, mechanism. Generally, primary structural similarity correlates well with the tertiary structure, and if proteins are truly homologous, then they are likely to have very similar three-dimensional structures (Saier 1999Citation ). Therefore, identification of ENT orthologs in simpler organisms provides the opportunity of using alternate experimental models in the study of various aspects of ENT protein structure and function. In this article, we present evidence of ENT orthologs in a number of experimentally accessible model organisms including prokaryotes (which are widely used for structural studies on membrane proteins) and fish (which are attractive models for genetic manipulation, and developmental and functional studies of vertebrate proteins). We also present novel ENT family members in humans and mice.

The origin of the ENT family has always been elusive, and they have been assumed to be absent in prokaryotes. However, approximately 10% of genes in bacteria encode transport proteins (Driessen, Rosen, and Konings 2000Citation ), and many transporters found in eukaryotes have prokaryotic homologs (Blackmore, McNaughton, and van Veen 2001Citation ; Yen et al. 2002Citation ). Because equilibrative transport is likely to be an ancient function, it seemed possible to us that a prokaryotic ENT family member existed, although it would be relatively "hard to see" based on sequence similarity, given the extensive time since divergence of the three major domains (bacteria, archaea, and eukarya) of life (Feng, Cho, and Doolittle 1997Citation ). Sequence divergence can lead to proteins that are homologous (i.e., derived from a common ancestor) but that are unrecognizably different and have no statistically significant sequence similarity (Saier 1999Citation ). However, based on our analyses in combination with previous functional characterization of these proteins (Bremer et al. 1990Citation ; Nieweg and Bremer 1997Citation ), we propose that the Tsx family members are homologs of the eukaryotic ENTs.

The Tsx proteins have been identified in a number of gram-negative bacteria (Nieweg and Bremer 1997Citation ). These bacteria possess an outer membrane, the thickness of which is similar to that of other biological membranes. However, this outer membrane differs from eukaryotic membranes, in that it consists of an asymmetrical bilayer of phospholipid and lipopolysaccharide, producing a somewhat different context for transport proteins. Tsx proteins have been shown to transport nucleosides and deoxynucleosides (used as nutrients) by facilitative diffusion from the extracellular environment, where they are present at submicromolar concentrations (Hantke 1976Citation ; Krieger-Brauer and Braun 1980Citation ). Reconstitution of purified Tsx shows that it is a channel-forming protein with a nucleoside-specific binding site that can discriminate between compounds that are closely related in structure (Benz et al. 1988Citation ; Maier et al. 1988Citation ; Fsihi, Kottwitz, and Bremer 1993Citation ). Like many (though not all) functionally characterized eukaryotic nucleoside transporters (e.g., hENT1), Tsx proteins do not transport nucleobases or phosphorylated derivatives of nucleosides, and their predicted topology is an integral, multitransmembrane protein with a large periplasmic loop in the central portion of the protein which is suggestive of the prototypic ENT structure. Most strikingly, there is both sequence homology and conservation of specific residues in Tsx and eukaryotic ENTs (Region 2, fig. 3b ) in an area that has been implicated in substrate translocation (Hyde et al. 2001Citation ). In addition, the highly conserved glycines in this region have recently been demonstrated to be functionally important in both trafficking and uptake by hENT1 (SenGupta et al. 2002Citation ). We cannot determine, at this time, if Tsx is mechanistically homologous to eukaryotic ENTs, although we are currently pursuing structural characterization of the protein. The translocation mechanism of other specific outer membrane proteins, such as maltoporin, has been well described (Hofnung 1995Citation ).

The reasons underlying the absence of ENT homologs in the nonproteobacteria and archae is not clear. ENTs could be a late acquisition by this prokaryotic lineage or the other lineages may have "lost" their ENTs, relying instead on proton-dependent nucleoside transport mediated by concentrative nucleoside transporter (CNT) homologs (e.g., nupC in E. coli; Craig, Zhang, and Gallagher 1994Citation ). CNTs are present in the inner membrane of prokaryotes, including the gram-negative bacteria, suggesting that the ENTs are only required for the transport of nucleosides across the outer membrane of this group and therefore would not necessarily be present or functional in other prokaryotes.

In addition to the prokaryotic ENTs, the base of the phylogenetic tree is filled with other ENT proteins with diverse characteristics. In general, the Tsx proteins show more similarity with the alveolate, apicomplexan, protozoan parasite ENTs, TgAT, and PfENT1, which are themselves somewhat distinct, often showing the least conservation in regions 1–4, relative to the rest of the ENTs. Indeed, although they are all parasitic protozoans, there is a considerable separation between the lineage leading to the Tg-Pf ENTs compared with the Lm-Tb ENTs. This is consistent with other sequence data, suggesting a considerable phylogenetic distance between these groups (Baldauf and Doolittle 1997Citation ). In addition, parasites are typically subject to different selective pressures compared with nonparasitic organisms, and this has likely resulted in both sequence variability and functional diversity in the protozoan parasite ENTs which have been well-described by others (Carter, Landfear, and Ullman 2001Citation ). In addition, at least one parasitic protozoan ENT, TbNT2, is a proton symporter (Sanchez et al. 1999Citation ; Carter, Landfear, and Ullman 2001Citation ). This unusual feature is mirrored in the one plant ENT, AtENT1, to have been characterized (Mohlmann et al. 2001Citation ). The origin and physiological relevance of proton-dependent transport by these ENT proteins remains to be fully explained. The identification of a single, partial Dictyostelium ENT homolog demonstrates the existence of ENTs within the slime molds (Mycetozoa). The evolution of this taxon has been debated, but it now appears that they are a monophyletic group of more recent eukaryotes with a closer association to an animal-fungal grouping than to green plants or protists (Baldauf and Doolittle 1997Citation ). Although the Dictyostelium homolog, DdENT, appears to be a partial clone, our data are consistent with this interpretation because the sequence aligns toward the base of the phylogenetic tree but clearly with a leaning toward the invertebrate sequences rather than the protists or Arabidopsis.

Local, taxon-specific gene duplication events appear to be primarily (but possibly not exclusively) responsible for the presence of homologs (paralogs) in the protists and plants. However, the presence of the ENT4 isoforms in the vertebrates that align or cluster more closely with invertebrate sequences than with other vertebrates sequences suggests that a duplication of the ENT lineage may have occurred, possibly around the time of the metazoan radiation, when a large increase in genetic diversity is proposed to have taken place (Graur and Li 2000Citation ). None of the members assigned to this lineage (h/mENT4, DmENT3, CeENT6) have been functionally characterized; therefore, nothing is known about the function and physiology of these proteins. Therefore, we speculate that duplication of an ancestral ENT may have led to two divergent lineages which ultimately gave rise to the ENT4 and ENT1/2/3 families in vertebrates.

Taxon-specific duplications in Drosophila and Caenorhabditis elegans appear to be primarily responsible for the additional homologs in these groups (Sankar et al. 2002Citation ). Although a comparative genomic analysis of the ENT family in C. elegans, Drosophila, and humans was recently conducted (Sankar et al. 2002Citation ), the complete functional characterization of any invertebrate ENT has not been reported in the literature. Both C. elegans and Drosophila represent attractive model organisms for a variety of genetic, localization, and physiological studies and will prove invaluable in advancing our understanding of ENT structure and function.

The tunicate or sea squirt, Ciona intestinalis, is another attractive model organism which has been used widely in developmental studies and in discerning the evolutionary origins of the chordate lineage (which subsequently led to the vertebrates). Ciona intestinalis is considered to be a presumptive ancestral chordate and has a small genome (relative to other chordates). Other transporters, such as the acetylcholine vesicular transporter (Takamura et al. 2002Citation ), have been isolated from C. intestinalis and show strong homology to their vertebrate counterparts. The presence of CiENT partial sequences that cluster with both the ENT1/2 lineage and the ENT3 lineage is suggestive of a possible additional gene duplication event occurring around, or after, the time of appearance of the vertebrates. This is a transitional period which is proposed to have been accompanied by a considerable increase in genetic diversity, possibly as a consequence of either one or two complete genome duplications, although this view is controversial (Ohno 1999Citation ; Graur and Li 2000Citation ; Friedman and Hughes 2001Citation ). The CiENT sequences we have identified align with different regions of the other ENTs, so we cannot confirm whether we have two different homologs or two different parts of a single homolog which are equally similar to both ENT lineages. In addition, we have yet to identify a CiENT4-type protein. Further characterization of the tunicate sequences and completion of the Ciona genome sequencing project may help clarify ENT evolution in the early vertebrates.

The presence of at least four teleost ENTs suggests that orthologs of ENT1–4 exist in this group and would support the concept of an ENT1/2 split being before or around the time of the appearance of the vertebrates. In addition to possible genome duplication in an ancestral vertebrate, an ancestral teleost is also speculated to have undergone a genome duplication (resulting in rapid evolution of this taxon), leading to the existence of multiple copies of orthologs compared with mammals (Taylor et al. 2001Citation ). This would suggest that more ENTs may exist in Fugu rubripes and may explain the presence of the additional partial sequences, although a full analysis of the ENT history in the teleosts awaits completion of either the pufferfish or zebrafish genome sequencing project (or both).

In addition to novel ENT homologs in teleosts, urochordata, mycetozoa, and prokaryotes, we have identified novel family members (mENT4, hENT4) in mammals. This unexpected finding suggests considerably greater complexity in the cellular physiology of ENTs (and their role in the uptake and efficacy of nucleoside analog drugs) than previously determined based on pharmacological data (which predicted only two ENT types). Intracellular nucleoside uptake has been described (Pisoni and Thoene 1989Citation ), and ENT3 has been proposed as an intracellular ENT because it has putative dileucine targeting motifs in the N-terminus (Hyde et al. 2001Citation ). We could not identify similar motifs (dileucine repeats, lysosomal targeting signals) in m/hENT4, but these sequences are often cryptic, and further analysis may indicate a putative location (possibly intracellular) for these proteins.

We have identified a number of regions that are highly conserved across all taxa. Within these regions are conserved residues and motifs. In some cases, functional investigation of these regions has already begun, such that region 1 has been implicated as being part of the protein that interacts with nonnucleoside analog inhibitors because mutation of residue 33 in hENT1 and hENT2 alters their sensitivities to these compounds (Visser et al. 2002aCitation ). This residue is typically a hydrophobic residue in all the other ENTs, but a highly conserved motif (GXGS/TXXPW/YNXXX, where X is typically a hydrophobic residue) precedes this position, suggesting that this overall region is functionally important. Interestingly, asparagine (N) 338 in hENT1 has also been found to be critical for interaction with high-affinity inhibitors (Visser et al. 2002bCitation ). N338 is found within region 3 and this residue is conserved in 42 ENTs, including the bacterial Tsx proteins, where it forms part of a motif (N-XXDXXGR, where X is generally hydrophobic). The functional role of this region is under investigation (Visser et al. 2002bCitation ), but it may be part of a recognition domain, possibly in combination with other parts of the ENT protein.

The presence of conserved motifs implicated in translocation, and possibly recognition, in Tsx suggests that this may be a useful model for further structural studies on ENTs. A number of other conserved residues and motifs remain to be investigated such as those found in region 4. To our knowledge, at this time, there are no data on the functional significance of the conserved arginine and proline residues or the conserved S/TNGY motif found in this region, and these patterns have not been identified as being significant in other proteins.

We have identified a number of novel ENTs in phylogenetically diverse and experimentally important organisms. This greatly increases our understanding of the distribution and origins of this multiisoform family and provides insights into regions of the protein that have been conserved during a long evolutionary history. We have proposed a scenario for the evolution of the ENTs, not as a conclusive account, but rather as a starting point for further analyses and studies which we anticipate will lead to additional growth, budding, and branching of the ENT family tree.


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A corrected version of hENT4 mRNA and protein, which removes the portion of the protein resembling phosphorylase kinase, has now been entered into GenBank under the accession number gi: 22062464. Nucleotide sequence data and a complete annotated gene sequence for hENT4 are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank databases under the accession number TPA: BK000627.


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We thank Dr. Barrie Coukell, York University, for bringing the Dictyostelium sequence to our attention, Dr. James Coulton, McGill University, for bringing the Tsx proteins to our attention, and Dr. Laurence Packer, York University, for useful discussions. The data on the fish putative ENTs have been provided freely by the Fugu Genome Consortium for use in this publication only. This work was supported by grants from the Natural Science and Engineering Research Council (NSERC—#203397–98) and National Cancer Institutes (NCIC #011070) of Canada to I.R.C.


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Brian Golding, Reviewing Editor

Keywords: equilibrative nucleoside transporters prokaryotes evolution ENT4 Back

Address for correspondence and reprints: Imogen R. Coe, Department of Biology, York University, 4700 Keele St., Toronto, Canada M3J 1P3. E-mail: coe{at}yorku.ca . Back


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Accepted for publication August 11, 2002.