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J. Biol. Chem., Vol. 277, Issue 24, 21499-21504, June 14, 2002

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Six Related Nucleoside/Nucleobase Transporters from Trypanosoma brucei Exhibit Distinct Biochemical Functions^*

Marco A. Sanchez, Rob Tryon, Joy Green, Ilja Boor, and Scott M. Landfear^§

From the Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon 97201

Received for publication, March 9, 2002, and in revised form, April 2, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 µM. 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

African trypanosomes are of considerable medical and economic importance because they cause a debilitating disease in humans (sleeping sickness) and livestock (nagana) throughout a large portion of sub-Saharan Africa (1). These parasites have a digenetic life cycle, with two main stages: the bloodstream form (BF)¹ that lives in the bloodstream of its mammalian host and the procyclic form (PF) that lives in the insect vector (tsetse fly). Purines are essential for the growth, multiplication, and survival of these organisms because the parasites are incapable of synthesizing the purine ring de novo (2, 3). Furthermore, nucleoside/nucleobase transporters are of considerable pharmacological importance, because both purine analogs and non-purine analog drugs are taken up by some of these permeases, and loss of permease function can lead to drug resistance (4, 5).

Two different nucleoside transport systems have been characterized in intact Trypanosoma brucei cells. The P1 type system mediates the uptake of purine nucleosides (adenosine, inosine, and guanosine) and is detected in both BF and PF life cycle stages, and the P2 type system mediates the uptake of adenosine and adenine, as well as several anti-trypanosomal drugs, and is detected only in the BF (6, 7) parasites. In addition, four nucleobase transport activities have also been identified. H1, H2, and H3 mediate the transport of hypoxanthine, guanine, and adenine (8, 9). H1 activity is found in PF, and H2 and H3 activities are found in BF. In addition, the U1 activity mediates the transport of uracil in PFs (10). However, meticulous functional and biochemical characterization of these transporters at the molecular level is needed to understand the biological role of purine transporters in survival and adaptation of T. brucei to different environments during its life cycle and to provide information about drug delivery and drug resistance phenotypes associated with purine transporters (11).

Previously we cloned and characterized the T. brucei nucleoside transporter 2 gene, TbNT2, which encodes a P1 type transporter expressed only in the BFs (12). In this study we demonstrate that TbNT2 is a member of a multigene family. In the reference strain TREU 927 used for the T. brucei genome project (parsun1.path.cam.ac.uk/index.html), this family encodes six similar but distinct transporters, denominated TbNT2/927 to TbNT7 (the designation TbNT2/927 is used to distinguish this gene from the closely related but nonetheless distinct TbNT2 gene that was derived originally from T. brucei strain EATRO 110 (12)). All of the family members are clustered together in chromosome number II and are separated by ~9-kb intergenic regions. Functional expression of TbNT2/927 through TbNT7 in Xenopus oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 transport purine nucleosides with similar affinities in the low micromolar range. Of note, TbNT5, and to a lesser extent TbNT6 and TbNT7, also show significant hypoxanthine transport activity. Moreover, ribonuclease protection assays indicate that the mRNAs from all six genes are expressed in BF parasites, but only TbNT2/927 and TbNT5 mRNAs are expressed at detectable levels in both BFs and PFs. Consequently, members of this nucleoside transporter family are differentially regulated during the parasite life cycle and mediate the uptake of purine nucleosides and in some cases also the nucleobase hypoxanthine. The presence of several P1 type transporters with similar but distinct biochemical properties and divergent regulation of expression suggests that the purine transport process in T. brucei is much more complex than was assumed on the basis of previous studies with whole parasites.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals-- [-³²P]dCTP (3000 Ci mmol¹), [-³²P]UTP (800 Ci mmol¹), [2,8,5'-³H]adenosine (54.4 Ci mmol¹), and [2,8-³H]adenine (28.8 Ci mmol¹) were purchased from PerkinElmer Life Sciences; [2,8-³H]inosine (34 Ci mmol¹) and [8-³H]guanine (15 Ci mmol¹) were purchased from American Radiolabeled Chemicals Inc.; and [2,8-³H]hypoxanthine (24.5 Ci mmol¹) and [8-³H]guanosine (5 Ci mmol¹) were purchased from Movarek Biochemicals. All of the other chemicals were of the highest commercial quality available.

Growth of Parasites and Isolation of Nucleic Acids-- Procyclic forms of T. brucei strain TREU 927 (13) were grown at 26 °C in Cunningham's medium (14). The bloodstream forms of T. brucei strain TREU 927 from frozen stocks were grown in Wistar rats, and blood was collected through exsanguination. BFs were separated from blood cells on a DE52 (Whatman) anion exchange column as described (15). The nucleic acids were purified from trypanosomes following established procedures (16), and Southern blots were performed using standard protocols (16).

Cloning and Sequencing of TbNT Family Members-- An RPCI-93 BAC (library 93 made at Roswell Park Cancer Institute, School of Medicine, Buffalo, NY) clone designated 36E18 (EMBL/GenBank^TM accession number AC007866) containing the TbNT family cluster was identified by a BLAST search (17) of the T. brucei data base (www2.ebi.ac.uk/blast2/parasites.html) using the TbNT2 amino acid sequence (12) as a query sequence. To subclone each gene from the BAC clone 36E18, 100 ng of BAC DNA was used as template for six independent PCR amplifications. The oligonucleotide O1 (5'-GGGGTACCACCATGGCAATGCTTGGT-3'), representing the first five identical amino acids of the TbNT family including a KpnI restriction site (underlined) and a consensus Kozak sequence (18) (italics), was used as forward primer, and a specific oligonucleotide, representing the complement of the sequence within the 3'-untranslated region of each TbNT ORF, was used as reverse primer. PCR amplification was performed using Pfu Turbo^TM polymerase (Stratagene) following the manufacturer's instructions. Amplified DNA fragments were subcloned using the Zero Blunt^TM TOPO PCR cloning kit (Invitrogen). The clones were further characterized by restriction mapping and sequencing of the entire ORF. Oligonucleotide synthesis and automatic sequencing were performed by the Core Facility of the Department of Molecular Microbiology and Immunology at the Oregon Health and Science University using a model 394 DNA/RNA synthesizer (Applied Biosystems) and the ABI model 377 DNA sequencer (PerkinElmer Life Sciences).

DNA and Deduced Amino Acid Sequence Analysis-- DNA and deduced amino acid sequence analysis were performed by using the MacVector software (Intelligenetics). Transmembrane segments were predicted using the TMPRED software (www.ch.embnet.org/software/TMPRED_form.html).

TbNT2/927 Gene Family Expression in Xenopus Oocytes-- The TbNT2/927 gene family ORFs were subcloned into the EcoRI site of the Xenopus expression vector pL2-5 (19), linearized, and in vitro transcribed with T7 RNA polymerase (Invitrogen) in the presence of CAP analog (Amersham Biosciences) as described previously (20). Stage V-VI Xenopus oocytes were injected with 23 nl of cRNA (~10 ng), incubated in ND96 buffer for 3 days at 16 °C as described (20), and used for uptake assays.

Uptake Assays-- Xenopus oocytes injected with cRNA or water as control were incubated for 3 days after injection. Prior to the assay, the oocytes were incubated for 30 min in ND96 buffer at room temperature. Uptake of [³H]adenosine, [³H]inosine, [³H]guanosine, [³H]hypoxanthine, [³H]guanine, and [³H]adenine was assayed by incubating oocytes with radiolabeled substrates for the indicated times, followed by three quick washes in cold ND96 buffer, and the samples were prepared for liquid scintillation counting as described previously (12). For each data point, the number of picomoles of labeled substrate transported were calculated and plotted as a function of incubation time. These data were fitted to a straight line by a linear regression analysis with CA-Cricket Graph III software (Computer Associates International Inc.). To perform substrate saturation curves, cRNA-injected oocytes were incubated for 60 min in the presence of different concentrations of substrate at room temperature. Control experiments demonstrated that the uptake of substrate was linear over 60 min over the range of concentrations tested. The K_m values were estimated by fitting the substrate saturation curves to the Michaelis-Menten equation by nonlinear regression using the KaleidaGraph program (Synergy Software).

Ribonuclease Protection Assay-- To explore the expression of the TbNT2/927 family members, a ribonuclease protection assay was performed by using the RPA III^TM ribonuclease protection assay kit (Ambion), following the protocols recommended by the manufacturer. Briefly, 10 µg of total RNA from TREU 927 PF or BF were hybridized against specific antisense ³²P-labeled riboprobes, generated by the MAXIscript^TM T7 in vitro transcription kit (Ambion), representing the complement of the sequence within the 3'-untranslated region of each TbNT ~150 bp downstream from the 3' end of each ORF. -Tubulin was used as a control gene that is expressed at similar levels in BFs and PFs. After RNase A/T1 treatment and ethanol precipitation, protected RNA was resolved on 6% denaturing polyacrylamide gel and detected by exposing the gel to x-ray film (Kodak OMAT-AR) or to a PhosphorImager screen.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The TbNT2/927 Gene Family Encodes P1 Type Nucleoside Transporter Isoforms-- Biochemical characterization of purine nucleoside transport in intact T. brucei parasites indicated the presence of P1 type transport that mediates the uptake of adenosine and inosine in both PFs and BFs (6). In previous studies (12), we have cloned a P1 type transporter gene from T. brucei strain EATRO 110, called TbNT2, that is a high affinity adenosine/inosine transporter whose RNA is detectable only in BFs. Genomic Southern blots hybridized with the TbNT2 ORF indicated the presence of a multigene family, raising the possibility that some of these TbNT2-like genes might encode other P1 type transporters.

To clone TbNT2-like genes, we first BLAST searched the T. brucei genome data base using the TbNT2 amino acid sequence as a query, and six ORFs were identified. All of the six ORFs were contained in the same RPCI-93 BAC clone 36E18, which contains a genomic DNA insert from chromosome number II of T. brucei strain TREU 927 (www.tigr.org/tdb/mdb/tbdb/progress.html), and the predicted amino acid sequences showed high identity to TbNT2 (81-96% identity). The gene that predicted a protein with the highest identity (96%) to the original TbNT2 was designated TbNT2/927 (T. brucei nucleoside transporter 2/927). Moreover, TbNT2/927 is the first gene in the array of the locus according to the predicted transcription direction (Fig. 1). The other P1 type genes were designated TbNT3 through TbNT7. All genes were cloned using a PCR strategy and sequenced to determine the identity of each one by comparing the DNA and predicted amino acid sequences with those obtained from the T. brucei data base. In addition, we identified five copies of an interspersed unrelated gene within the TbNT array, encoding a putative isopenicillium-N-synthase gene (INS in Fig. 1).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the TbNT2/927 gene family in T. brucei TREU 927 strain. The complete BAC 36E18 DNA sequence containing the TbNT2/927 gene family cluster was retrieved from the GenBankTM (accession number AC007866). The arrows represent the TbNT ORFs, the black boxes represent the isopenicillium synthase (INS) homolog, the thick black lines indicate the intergenic regions, and the diagnostic restriction enzyme sites are indicated. The representation is not drawn to scale. In the numbering scheme employed, TbNT7 is positioned to the left of TbNT6. The reason for this nonsequential numbering is that several ORFs were identified, named, and partially characterized before the physical order of the ORFs had been determined by assembly of the entire 36E18 BAC clone sequence.

Gene multiplicity is a common feature in kinetoplastid protozoa (21), and each gene often encodes the same protein (22). However, in some examples, gene families encode similar but not identical proteins (23, 24). The six predicted TbNT proteins were very similar along the entire amino acid sequences (Fig. 2), suggesting that they are nucleoside transporter isoforms. Moreover, the predicted topology indicated 11 TMDs for all six proteins, similar to the human equilibrative nucleoside transporter 1 (ENT1), whose topology was recently experimentally elucidated by Sundaram et al. (25). The 11 TMDs shown in Fig. 2 are those predicted for TbNT2/927. The overall comparison showed four regions of major divergence between the six protein sequences. Those regions are limited to the extracellular loops between TMD1 and TMD2, TMD5 and TMD6, and TMD7 and TMD8 and the large intracellular loop between TMD6 and TMD7.

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Multi-alignment of the deduced amino acid sequence of TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7. Alignment was performed using CLUSTALW (MacVector, Intelligenetics). The spaces introduced to optimize the alignment are indicated by periods. Labeled solid lines over the TbNT2/927 sequence indicate the predicted transmembrane domains (www.ch.embnet.org/software/TMPRED_form.html) for this protein. The numbers at the right indicate the amino acid positions in each sequence.

Functional Expression of TbNT2/927 through TbNT7 in Xenopus Oocytes-- To identify the substrate specificity of the TbNT2/927 family members, we tested the ability of oocytes expressing TbNT2/927 through TbNT7 to mediate the uptake of purine nucleosides. Initial experiments indicated that TbNT2/927, TbNT5, TbNT6, and TbNT7 cRNA-injected oocytes were able to transport adenosine, inosine, and guanosine at significantly higher rates than the control water-injected oocytes (Fig. 3A). However, TbNT3 and TbNT4 cRNA-injected oocytes did not mediate the uptake of any purine nucleosides compared with the control water-injected oocytes (data not shown). To characterize the affinity of these transporters for adenosine and inosine, the K_m values were calculated from substrate saturation curves (Fig. 3B), revealing that TbNT2/927, TbNT5, TbNT6, and TbNT7 were high affinity adenosine/inosine transporters (Table I) with K_m values of <5 µM.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Functional expression of cRNA in Xenopus laevis oocytes. A, time course for uptake of 0.5 µM [3H]adenosine (Ado) by oocytes injected with TbNT5 cRNA (closed circles) or by oocytes injected with water (open circles) as control. For each time point, uptake (pmol) into at least three oocytes was measured and averaged; the error bars represent the standard deviations of these values. B, substrate saturation curve for [3H]adenosine in oocytes injected with TbNT5. For each [3H]adenosine concentration, at least three oocytes were incubated with the substrate for 60 min, and the individual velocities were averaged; the error bars represent the standard deviations of these values.

To test for potential functional differences between the distinct proteins, we also examined the ability of TbNT2/927 through TbNT7 to mediate the transport of purine nucleobases. Interestingly TbNT5 cRNA-injected oocytes were able to mediate the uptake of hypoxanthine at a significantly higher level than the control water-injected oocytes (Fig. 4). Moreover, saturation curves for TbNT5 revealed a K_m value of 49.4 ± 13.3 µM (mean ± S.D., n = 3) for hypoxanthine. In contrast TbNT6 and TbNT7 showed limited but still significant hypoxanthine transport activity, whereas TbNT2/927, TbNT3, and TbNT4 did not show any ability to transport this nucleobase. Additional experiments were conducted to test the transport of guanine and adenine, but none of the six permeases transported these purines (data not shown). In summary, there are clear differences in substrate specificity among members of the TbNT2/927 family. TbNT2/927 is a purine nucleoside transporter, TbNT5, TbNT6, and TbNT7 transport hypoxanthine in addition to the purine nucleosides, and TbNT3 and TbNT4 have not exhibited any clear transport activity for the substrates tested here including adenosine, inosine, guanosine, xanthosine, adenine, hypoxanthine, guanine, xanthine, cytosine, uracil, thymine, cytidine, thymidine, uridine, S-adenosylmethionine, spermidine, putrescine, and AMP.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   TbNT2/927 family shows differences in substrate specificity. Uptake assays were performed for 60 min in the presence of 0.5 µM [3H]adenosine (empty bars) or 25 µM [3H]hypoxanthine (filled bars) in TbNT2/927 through TbNT7 cRNA-injected oocytes. Each bar represents the average of at least three independent measurements, and the error bars indicate the standard deviations. The asterisks indicate values that are significantly different (p < 0.02) from the control water-injected oocytes as determined by the two-tailed Student t test. Note that uptake of hypoxanthine in control water-injected oocytes (2 ± 0.3 pmol oocyte1 h1) is considerably higher than uptake of adenosine (0.22 ± 0.04 pmol oocyte1 h1).

In intact T. brucei parasites, transport of nucleosides (7) and nucleobases (8) has been shown to be dependent upon the transmembrane proton motive force, strongly suggesting that these permeases are active proton symporters. Indeed, uptake of adenosine by TbNT2/927, TbNT5, TbNT6, and TbNT7 cRNA-injected oocytes was significantly inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol (data not shown), suggesting that the adenosine uptake by these permeases is coupled to proton translocation.

TbNT2/927 Gene Family Expression in the Life Cycle of T. brucei-- To explore the expression of each member of the TbNT2/927 family in the two major life cycle stages of T. brucei, a ribonuclease protection assay was performed employing specific antisense ³²P-labeled riboprobes from the 3'-untranslated region of each gene. Ribonuclease protection assay experiments revealed that RNA from all of the members of the family was detected in the BFs (Fig. 5). In addition, RNA from TbNT2/927 and TbNT5 was also detected in the PFs. However, TbNT2/927 expression was similar in both BF and PF life cycle stages, whereas TbNT5 expression was up-regulated in the BF stage. -Tubulin riboprobe was used as a control for an RNA that is expressed at similar levels in both the BF and PF life cycle stages of T. brucei.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   TbNT2/927 gene family is differentially expressed during the life cycle of T. brucei. Fragments protected from RNase digestion by specific antisense riboprobes were resolved in 6% acrylamide, 8 M urea gels and exposed to x-ray films or to a PhosphorImager screen. BF lanes indicate protected fragments using RNA from bloodstream form parasites, and PF lanes indicate protected fragments using RNA from procyclic form parasites. A control -tubulin (-tub) riboprobe was used to demonstrate that similar amounts of PF and BF mRNA were present in each sample.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In T. brucei, purine nucleoside and nucleobase transporters have been extensively studied at the biochemical level (6, 8, 9) and shown to play important roles in the biochemistry and pharmacology of these parasites. More recently, studies at the molecular level began with the cloning of the TbAT1 (P2 type nucleoside transporter) (26) and of the TbNT2 (a P1 type nucleoside transporter) (12) genes. Thus, TbAT1 and TbNT2 function correlated with many of the biochemical features of nucleoside transport in intact parasites. However, the original TbNT2 gene (EATRO 110 strain) encoding a P1 type transporter was expressed only in BF parasites, whereas P1 activity could be detected in both BFs and PFs (6). These results, along with the observation of multiple fragments on genomic Southern blots that hybridized to a TbNT2 probe (12), suggested that other P1 type transporters were likely to exist. In this study, we report the functional expression and characterization of the TbNT2/927 gene family, four members of which encode high affinity adenosine, inosine, guanosine transporters, placing them within the P1 type of nucleoside transporters previously defined at the biochemical level (6, 12). However, TbNT5, TbNT6, and TbNT7 were also able to mediate the uptake of hypoxanthine, a nucleobase that plays a central role in the purine salvage pathway of these parasites (3). This result is consistent with previous observations that some other ENT family members, specifically TbAT1 (26), human ENT2 (27), and PfNT1 (28), are able to transport some nucleobases, most often with substantially higher K_m values than for nucleosides.

It will be interesting to determine what specific molecular determinants confer hypoxanthine transport function upon TbNT5, TbNT6, and TbNT7. Examination of the multi-alignment (Fig. 2) reveals that there are five amino acids (Val⁴⁷, Lys⁴⁸, Lys⁵², Pro⁵⁵, and Val⁷⁸ in TbNT5) that are conserved in these three permeases but that are different in TbNT2/927, TbNT3, and TbNT4. The first four of these residues are located in the extracellular loop between predicted TMD1 and TMD2, and Val⁷⁸ is located within predicted TMD2. It is possible that some of these residues confer hypoxanthine transport capacity upon TbNT5, TbNT6, and TbNT7.

It is noteworthy that we have not been able to identify any substrates for TbNT3 or TbNT4 in the Xenopus oocytes expression system. One possibility is that TbNT3 and TbNT4 are not functional transporters. Alternatively, the two permeases may transport some substrate that we have not tested. Compounds that we have examined as potential substrates for TbNT3 and TbNT4 include purine nucleosides and nucleobases, pyrimidine nucleosides and nucleobases, S-adenosylmethionine, polyamines, and AMP, but none of these solutes are substrates. We have also considered the possibility that TbNT3 and TbNT4 could form functional hetero-oligomers. However, coinjection of TbNT3 and TbNT4 cRNAs into oocytes did not elicit any transport function for any of the compounds listed above. Still another possible explanation for the failure of these proteins to mediate purine transport is that they require for function other subunits that are not present in the oocytes. The requirement for multiple subunits has been observed for several amino acid transporters (29).

There is ample precedent for the existence of multiple isoforms of various transporters in both unicellular and multicellular eukaryotes (30, 31). In multicellular organisms, distinct isoforms may be expressed in different tissues where they subserve the physiological needs of each cell type (32). The TbNT2/927 family encompasses six permeases whose sequences are distinct but closely related. It is possible that each permease has unique properties that collectively promote the viability of the parasite in its natural environment. Thus, examination of substrate specificity for each permease has revealed differences that could in part explain the distinct roles of different family members. Furthermore all six mRNAs are expressed in BF parasites, but only TbNT2 and TbNT5 mRNAs are also expressed in PF parasites. This observation raises the possibility that different purine permeases could be differentially regulated to accommodate the potentially distinct nucleoside composition of the mammalian bloodstream and the tsetse fly gut. Finally, it is possible that different members of this family are targeted to discrete subcellular locations where they might subserve unique functions. Ultimately, it will be important to define the potentially unique biological roles of each member of the TbNT2/927 family.

A substantial amount of the T. brucei genome has been sequenced as genome survey sequence clones (www.sanger.ac.uk/Projects/T_brucei/), raising the likelihood that many of the T. brucei genes encoding nucleoside or nucleobase transporters that are members of the ENT family have been at least partially sequenced and can be identified by BLAST searches. Using such "data base mining" approaches, we have identified two new ENT family members designated TbNT8 and TbNT9.² There are multiple TbNT8 genes arranged in a tandem cluster representing very closely related ORFs, whereas TbNT9 appears to be a single copy gene. Functional expression of both of these genes in Xenopus oocytes reveals transport activity for both nucleobases and nucleosides. However, we cannot yet rule out the possibility that other nucleoside/nucleobase transporters exist that exhibit low homology to the currently identified permeases or that are members of different transporter families.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AI 44138 (to S. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Rd., L220, Portland, OR 97201-3098. Tel.: 503-494-7588; Fax: 503-494-6862; E-mail: sanchezm@ohsu.edu.

^§ Recipient of the Burroughs Wellcome Fund Scholar Award in Molecular Parasitology.

Published, JBC Papers in Press, April 5, 2002, DOI 10.1074/jbc.M202319200

² M. A. Sanchez, C. Henriques, M. van Ampting, and S. M. Landfear, unpublished data.

	ABBREVIATIONS

The abbreviations used are: BF, bloodstream form; PF, procyclic form; ORF, open reading frame; ENT, equilibrative nucleoside transporter; TMD, transmembrane domain; BAC, bacterial artificial chromosome.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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