Equilibrative Nucleoside Transporters of Arabidopsis thalianacDNA CLONING, EXPRESSION PATTERN, AND ANALYSIS OF TRANSPORT ACTIVITIES*

Received for publication, May 7, 2003Published, JBC Papers in Press, June 16, 2003, DOI 10.1074/jbc.M304768200

Guangyong Li‡, Kunfan Liu‡, Stephen A. Baldwin§, and Daowen Wang‡¶

From the ‡Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, Chinaand the §School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

Equilibrative nucleoside transporters (ENTs) occur indiverse organisms. In the model plant Arabidopsis thali-ana, eight potential ENTs (AtENTs) have been predictedby genome sequencing. We here report the cloning of thecDNAs for AtENTs 2, 3, 4, 6, 7, and 8. Conceptual trans-lation of the cDNAs of AtENTs 2, 3, 4, 6, 7, and 8 yieldedpolypeptides possessing strong similarities to ENTscharacterized previously. Eleven putative transmem-brane domains were identified in each of the six At-ENTs. In suspension cells, the transcription of AtENTs 1,3, 4, 6, and 8 was increased by two treatments (nitrogendeprivation, application of 5-fluorouracil and metho-trexate) that inhibited the de novo pathway of nucleo-tide synthesis, indicating that multiple members of theArabidopsis ENT family may function in the salvagepathway of nucleotide synthesis. Except for AtENT1, thetranscription of the remaining six AtENTs showed vary-ing degrees of organ specificity. However, all seven At-ENTs were expressed in the leaf and flower. In plant,insect, and yeast cells, ectopically expressed AtENT3was targeted to the plasma membrane. AtENT3 ex-pressed in yeast cells transported adenosine and uri-dine with high affinity. Furthermore, the activities ofAtENT3 appear not to require a transmembrane protongradient because protonophores did not abolish adeno-sine or uridine transport. In competition experiments,the transport of [3H]adenosine by AtENT3 was most sig-nificantly inhibited by a number of different purine andpyrimidine nucleosides and 2�-deoxynucleosides, al-though certain nucleobases and nucleotides were alsofound to have some inhibitory effect. This indicates thatAtENT3 may possess broad substrate specificity. Aden-osine and uridine transport by AtENT3, although partlysensitive to the vasodilator drugs dilazep and dipyri-damole, was resistant to the nucleoside analogue nitro-benzylmercaptopurine ribonucleoside. We concludethat AtENT3 represents the first ei type ENT character-ized from higher plants. The potential functions of ENTsin the biology of A. thaliana are discussed.

Nucleoside transporters mediate the transport of nucleosidesand their analogues across cell membranes (1–6). Their trans-

port activities are important to the salvage pathway of nucle-otide synthesis (1–6). According to the mechanism of transport,nucleoside transporters have been divided into two families.The equilibrative nucleoside transporters (ENTs)1 generallytransport nucleosides down their concentration gradients (5–7); against this generality is the finding that several ENTsrecently characterized from kinetoplastid protozoans and thehigher plant Arabidopsis thaliana are probable proton sym-porters (8–13). The concentrative nucleoside transporters(CNTs) catalyze the transport of nucleosides against their con-centration gradients and are either sodium or proton symport-ers (5, 6, 14). Topologically, ENTs studied thus far have allbeen predicted to possess 11 transmembrane helices (TMs) (5,15, 16). In the studies on a human ENT (hENT1), evidence hasbeen obtained indicating that the amino- and carboxyl-termi-nal regions are located on the cytoplasmic and extracellularfaces of the membrane, respectively (16). The regions linkingthe TMs are all hydrophilic but differ considerably in their size.The large loop between TMs 1 and 2 is composed of �41residues and is glycosylated and extracellular (5, 7, 16). Thesecond large loop (between TMs 6 and 7) contains �66 aminoacid residues and is cytoplasmic (5, 16). From amino acid se-quence comparisons, it has been suggested that ENTs fromother organisms (including insects, nematodes, protozoa,yeasts, and higher plants) may adopt a topology similar to thatof hENT1 (16).

The biochemical properties of several ENTs have been inves-tigated extensively. In general, ENTs are broadly selective insubstrate specificity (5, 6). However, their transport processesare differentially inhibited by nucleoside analogues (such as ni-trobenzylmercaptopurine ribonucleoside (NBMPR)) or vasodila-tor drugs (e.g. dipyridamole, dilazep, and draflazine). For exam-ple, the es type ENTs are sensitive to inhibition by NBMPR,whereas the ei type ENTs are not (1, 2, 17). Whereas the es typetransporter from humans is potently inhibited by vasodilatordrugs, the corresponding transporters from rodents and the eitype transporters from both humans and rodents are much lesssensitive to such drugs (6, 18–22). Through molecular studies onmammalian ENTs, a region encompassing TMs 1–6 has beendeduced to be involved in substrate/inhibitor binding (23, 24).More recently, it has been found that the replacement of a glycineresidue (residue 154) in TM 4 of hENT1 by serine results inreduced NBMPR binding and that residue 33 in TM 1 of hENTs1 and 2 is an important determinant of vasodilator drug sensi-tivity (25, 26). The mutation of a single glycine residue (residue

* This work was supported by National Natural Science Foundationof China Grant 39770491 and Chinese Academy of Sciences GrantKSCX2-SW-304. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF426399(AtENT2), AF426400 (AtENT3), AF426401 (AtENT4), AF426402(AtENT6), AF426403 (AtENT7), and AY187030 (AtENT8).

¶ To whom correspondence should be addressed. Tel.: 86-10-64889380; Fax: 86-10-64854467; E-mail: dwwang@genetics.ac.cn ordaowenwang@hotmail.com.

1 The abbreviations used are: ENT, equilibrative nucleoside trans-porter; AGI, Arabidopsis genome initiative, AtENT, A. thaliana ENT;hENT, human ENT; CCCP, carbonyl cyanide m-chlorophenylhydra-zone; CNT, concentrative nucleoside transporter; DNP, dinitrophenol;NBMPR, nitrobenzylmercaptopurine ribonucleoside; RT, reverse tran-scription; TM, transmembrane; GFP, green fluorescent protein; EGFP,enhanced green fluorescent protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 37, Issue of September 12, pp. 35732–35742, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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179) in TM 5 alters both nucleoside transport activity and sen-sitivity to NBMPR in hENT1 (27).

In addition to playing a key role in the salvage pathway ofnucleotide synthesis, mammalian ENTs have been found toaffect many physiological processes including neurotransmis-sion and cardiovascular activity through the regulation ofadenosine concentrations (1–6). Clinically, ENTs are targets ofvasodilator drugs as well as routes for uptake of nucleosidedrugs used in treating cancers and viral infections (3). Mam-malian ENTs are found in a variety of tissues and cells, andevidence for the presence of more than one type of ENTs in thesame tissue has been obtained (28, 29).

Compared with mammals and parasites, progress in under-standing the expression, structure, and function of higher plantENTs has been slow. Although nucleoside transport processeswere first recorded in plants more than 20 years ago and havesince been discovered in several cell types (30–32), the corre-sponding nucleoside transporters have not been identified. Bytaking advantage of the genetic information from the genomesequencing project, researchers have recently started molecu-lar and biochemical studies of the ENTs encoded by the modelplant A. thaliana (13, 33). A total of eight genes encodingpotential ENTs have been predicted in Arabidopsis (5). Theirputative products (designated as AtENT1 to -8) form an inde-pendent branch in the phylogenetic tree constructed usingENTs from mammals, insects, nematodes, protozoa, yeasts,and plants (5). The gene encoding AtENT1 is expressed consti-tutively and in multiple tissues (33). Functional expression ofAtENT1 in yeast cells has also been achieved (13). It wasshown that, in intact yeast cells, AtENT1 catalyzed a proton-linked, high affinity adenosine transport and that the trans-port mediated by AtENT1 was resistant to inhibition by thenucleoside analog NBMPR and by the vasodilator drugs di-lazep and dipyridamole (13). In addition, it was found thaturidine, which is commonly transported by various types ofmammalian ENTs, might not be significantly transported byAtENT1 (13). These results suggest that at least some (if notall) of the AtENTs predicted by genome sequencing are likely tobe functional proteins. In this context, it would be important tostudy all members of the Arabidopsis ENT family in orderto gain a more comprehensive understanding of the function ofhigher plant ENTs.

In protozoan parasites and some mammalian cell types, theimportance of ENTs in the salvage pathway of nucleotide syn-thesis is indicated by the lack of the de novo pathway of nucle-otide synthesis (4, 34–36). In higher plants, evidence for theparticipation of ENTs in the salvage pathway has mainly comefrom observations that exogenously supplied nucleosides (suchas [3H]thymidine) and analogs (for example, bromodeoxyuri-dine) can be taken up and incorporated into DNA synthesis(37–40). So far there have been no investigations on whetherthe transcription of ENT genes or the activities of ENT proteinswould change in response to inhibition of the de novo pathwayof nucleotide synthesis in plant cells. Experiments of this kindare essential for not only providing molecular evidence on thefunction of ENTs in the salvage pathway of nucleotide synthe-sis but also yielding insights into the relationship between thetwo pathways of nucleotide synthesis in plant cells. Based onthe above discussions, we decided to conduct a more compre-hensive study of the ENTs of A. thaliana predicted by genomesequencing. Below, we report the cloning of the cDNAs forAtENTs 2, 3, 4, 6, 7, and 8, comparative analysis of the aminoacid sequences of AtENTs with those from other organisms,regulation of AtENT transcription by alterations in the de novopathway of nucleotide synthesis, transcription patterns ofseven AtENTs in various Arabidopsis organs, and functional

expression of AtENT3 in yeast cells. The potential role of ENTsin A. thaliana is discussed in light of our results and thosepublished previously.

EXPERIMENTAL PROCEDURES

Cloning of AtENT cDNAs and Comparative Analysis of Amino AcidSequences of AtENTs—The Col-1 ecotype of A. thaliana was usedthroughout this study. To amplify cDNAs for the complete coding re-gions of AtENTs 2, 3, 4, 6, 7, and 8 using RT-PCR, oligonucleotideprimers (Table I) were synthesized according to genomic sequence in-formation generated by the Arabidopsis genome initiative (AGI). TotalRNA samples were prepared from suspension cells, leaves, stems, roots,flowers, or immature siliques of A. thaliana as described previously(41). RT-PCR experiments using appropriate primers and enzymeswere conducted following published protocols (41). The expected cDNAfragments were cloned into the pGEM®-T Easy plasmid vector (Pro-mega). The inserts in the resultant positive clones were sequencedcommercially (TaKaRa) from both strands. The obtained cDNA se-quences were compared with those of predicted AtENT coding regionsusing various programs at the NCBI Web site (www.ncbi.nlm.nih.gov).

For calculating amino acid sequence identities using the softwareDNAstar (DNAstar Inc), the amino acid sequences of AtENTs 2, 3, 4, 6,7, and 8 were deduced from cloned cDNAs and compared with those ofAtENT1 and ENTs from other sources for which nucleoside transportactivities have been demonstrated (Table II) (6). To investigate phylo-genetic relationships of AtENTs to ENTs from other sources, the aminoacid sequences were aligned using the ClustalW program at the EBIWeb site (www.ebi.ac.uk/clustalw/index.html). The aligned sequenceswere converted into MEGA format, which was subsequently employedfor constructing phylogenetic trees at the MEGA 2 Web site (www.megasoftware.net) (42). The putative transmembrane domains ofAtENTs were predicted using the HMMTOP program (available on theWorld Wide Web at www.enzim.hu/hmmtop/server/hmmtop.cgi) (43)with manual adjustment aided by the comparison of the amino acidsequences of AtENTs with those of hENTs 1 and 2 (for which there iscurrently more structural information available).

Transcriptional Responses of AtENTs to Nitrogen Deprivation andDrug Treatment in Arabidopsis Suspension Cells Using Semiquantita-tive RT-PCR—For investigating transcriptional responses of AtENTs tonitrogen deprivation or drug treatment, a suspension cell culture ofA. thaliana was initiated and maintained as previously described (41,44). Suspension cells grown in nitrogen-sufficient liquid medium (con-taining 25 mM KNO3 and 1 mM (NH4)2SO4) were collected, washed, anddivided into two batches. Both batches of cells were resuspended innitrogen-deficient medium (devoid of any NO3

� and NH4�). For the first

batch of cells, a sample was immediately taken as the day 0 control.Further samples were taken 1, 3, and 5 days later. Control sampleswere prepared at identical time points from cells cultured in nitrogen-sufficient medium. The second batch of cells, which had been in nitro-gen-deficient medium for 5 days, was collected, washed, and dispersedinto nitrogen sufficient medium. Cell samples were then taken at 1, 3,and 5 days after nitrogen resumption. The strategies for preparingsuspension cells for drug treatment and sample collection were essen-tially similar to those described above except that nitrogen-sufficientmedium was used. The two drugs employed were 5-fluorouracil (2 �M,diluted from a 250 mM solution prepared in 1 M NH4OH) and metho-trexate (1 �M, diluted from a 50 mM solution prepared in 1 M NH4OH).Control samples were prepared from suspension cells cultured in theliquid medium lacking the two drugs. Total RNA was extracted from theharvested cell samples and was converted to cDNA by reverse tran-scription (41). The cDNA contents in all reverse transcription mixtureswere normalized by amplifying the transcripts of tubulin using theprimers TuF and TuR (Table I) (41). Evaluation of the transcript levelsof the seven AtENTs in the different cell samples by PCR using thenormalized cDNA mixtures and gene-specific primers (Table I) wasthen carried out as described previously (41).

Evaluation of Organ Specificities of AtENTs Transcription inA. thaliana—Root, stem, leaf, flower, and immature silique sampleswere prepared from 7-week-old A. thaliana plants grown under normalphysiological conditions. Total RNA was extracted from the five sam-ples and was transcribed into cDNA (41). The cDNA contents in thereverse transcription mixtures were normalized as described above.The transcript levels of the seven AtENTs in the five organs wereevaluated by PCR using the normalized cDNA samples and gene-specific primers (Table I).

Localization of AtENT3 Expressed in Insect, Yeast, or Plant Cells—For all DNA cloning experiments in this study, the methods described

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by Sambrook et al. (45) were generally followed. The correctness of eachcloning step was verified by restriction enzyme digestion and sequenc-ing through the cloning junction(s). To facilitate the construction of anAtENT3:GFP fusion cistron for insect or yeast cell expression, thevector pFB-GFP was created by ligating a BglII/NotI fragment, whichcontains the EGFP open reading frame and several restriction sites(SalI, ApaI, BamHI, and AgeI) from the pEGFP-1N plasmid (Clontech),to BamHI/NotI-digested pFastBac vector (Invitrogen). The unique SalIsite upstream of the EGFP open reading frame in pFB-GFP was thenemployed for accepting the AtENT3 open reading frame that was am-plified by PCR using primers 3F and 3R (Table I) and the high fidelitypolymerase ExTaq (TaKaRa). The resultant construct pFB-AtENT3:EGFP and the control construct (pFB-GFP) were expressed in insectcells using the Bac-to-BacTM baculovirus expression system (Invitrogen)following the instructions provided by the supplier. The expressing cellswere examined using a confocal microscope (FV500; Olympus) at 48 hpost-transfection. Optical sections were prepared for selected cells at athickness of 0.35 �m. For yeast cell expression, the construct pY-AtENT3:EGFP was created by ligating an EcoRI/XbaI fragment ofAtENT3:EGFP (derived from pFB-AtENT3:EGFP) to the pYES2 vector(Invitrogen) linearized with the same two enzymes. A control construct,pYEGFP, was prepared by removing the AtENT3 coding sequence frompYAtENT3:EGFP using SalI digestion and self-ligation of the resultantplasmid backbone. Induction of the expression of pYAtENT3:EGFP andpYEGFP in yeast cells was carried out following the instructions byInvitrogen. Expressing cells were examined using confocal microscopy.For transient expression in plant cells, the construct p35S-AtENT3:GFP was created by ligating the SalI fragment of AtENT3 (see above)into the SalI site upstream of the coding sequence of GFP in the vectorp35S-GFP (provided by Dr. Jinsong Zhang, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences). Plasmid DNA ofp35S-AtENT3:GFP or p35S-GFP was introduced into onion epidermalcells using a particle bombardment method as described by Silva andGoring (46). The expressing cells were examined and photographedusing confocal microscopy. In some experiments, the bombarded onionepidermal strips were left for 15 min on filter papers soaked with 0.8 M

mannitol before being examined. This osmotic shock treatment causeda partial separation of the plasma membrane from the cell wall, whichgave a higher resolution image of the association of the AtENT3:GFPfusion protein with the plasma membrane.

Adenosine Transport of AtENT3 Expressed in the BMA64–1A ade2Strain of Saccharomyces cerevisiae—Under normal conditions, S. cer-evisiae cells do not take up adenosine because of the lack of an endog-enous transport system for the nucleoside (11). An ectopically expressednucleoside transporter may enable S. cerevisiae to take up adenosine ifit is functional (11, 13). Previous investigators have shown that theW303 ade2 strain of S. cerevisiae (W303, Mat �, leu2–3, leu2–112,trp1–1, ura3–1, his3–11, his3–15, ade2–1, can1–100), which is defectivein adenine biosynthesis and forms red colonies on medium lackingadenine, can be employed for functional expression of AtENT1 (13).However, when we attempted to use this strain for functional expres-sion of AtENT3, many white colonies formed on medium containingadenosine. To avoid potential difficulties in using the W303 ade2 strain,we chose to perform functional expression of AtENTs in an alternativeade2 strain (BMA64–1A, Mat �, ura3–52, trp1�2, leu2–3-112, his3–11,ade2–1, can1–100, purchased from Euroscarf). To verify the suitabilityof the BMA64–1A ade2 strain, two AtENT1 constructs, pYAtENT1 andpYGFP:AtENT1, were prepared and expressed. To prepare pYAtENT1,a BamHI/EcoRI fragment of the AtENT1 open reading frame (33) wascloned into the pYES2 vector that had previously been cut with thesame two enzymes. pYGFP:AtENT1 was constructed by inserting aBamHI fragment of GFP (33) into the BamHI site of pYAtENT1. Twoconstructs, pYAtENT3 and pYAtENT3:EGFP, were used for functionalexpression of AtENT3 in the BMA64–1A ade2 strain. The preparationof pYAtENT3:EGFP was described above. pYAtENT3 was constructedby cloning the complete coding region of AtENT3, which was amplifiedby PCR using primers 3F and 3RS (Table I) and ExTaq, into the XhoIsite of pYES2. The four expression constructs (pYAtENT1, pYGFP:AtENT1, pYAtENT3, and pYAtENT3:EGFP) and the control plasmidpYES2 were each transformed into the cells of the BMA64–1A ade2strain. For plate assays, the cells were grown on three types of medium:minimal medium containing full complement of nutrients (as describedby Invitrogen), minimal medium lacking uracil, and minimal mediumlacking both uracil and adenine but containing 150 �M adenosine(Sigma).

The yeast cells expressing pYAtENT1 or pYAtENT3 were used foruptake experiments. Cells were cultured to an A600 of 0.7–1.5 in liquidmedium containing galactose (as described by Invitrogen), collected by

centrifugation, and washed twice with 25 mM phosphate buffer (pH 6.0).The washed cells were resuspended to an A600 of 3 in 25 mM phosphatebuffer and were kept on ice. For uptake assays, 100 �l of cells weregently mixed with an equal volume of transport medium (25 mM phos-phate buffer, pH 6.0) containing the desired concentrations of [3H]ade-nosine (Amersham Biosciences). The mixture was layered on top of 200�l of oil in an Eppendorf tube and was kept at 25 °C for the requiredtime (47). Uptake was terminated by a 2-min centrifugation at 12,000 �g. The supernatant (containing unincorporated [3H]adenosine) was re-moved by aspiration. The pelleted cells were washed twice with 25 mM

phosphate buffer and were solubilized with 5% Triton X-100 for meas-uring the level of [3H]adenosine uptake in a scintillation counter(MicroBeta Trilux; PerkinElmer Life Sciences). [3H]Adenosine uptakerate was expressed as pmol/mg of yeast protein. The quantification ofyeast protein was carried out using a Bio-Rad protein assay kit(Bio-Rad). Kinetic parameters (Km, Vmax) were calculated by nonlinearregression using the SigmaPlot 2000 software (SPSS Inc., Chicago, IL).For investigating the sensitivity of AtENT3-mediated transport to in-hibitors, vasodilator drug (dilazep or dipyridamole; Sigma), nucleosideanalog (NBMPR; Sigma), or protonophore (CCCP or DNP; Sigma) ofdesired concentrations were added to the yeast cell suspension (intransport medium) 30 min before the addition of [3H]adenosine and theinitiation of the uptake assay (26).

Uridine Transport of AtENT3 Expressed in the fui1 Strain ofS. cerevisiae—In S. cerevisiae, Fui1p is a cell surface uridine trans-porter (47). The mutation of its coding gene, fui1, could eliminateuridine influx into the yeast cells (47). Based on this property, a fui1knock-out strain, fui1:TRP1, has been used successfully for studyinguridine transport activities of hENT 1 and 2 (26, 48). To investigatepotential uridine transport activities of AtENT3, we expressed AtENT3cDNA in a similar fui1 knock out strain (BY4741, Mat �, his3�1,leu2�0, met15�0, ura3�0, YBL042C:KanMX4, purchased fromEuroscarf). The coding sequence of AtENT3 was amplified by PCRusing primers 3F2 and 3R2 (Table I). The amplified fragment wasdigested with the restriction enzymes EcoRI and NotI, followed bycloning into the p181AINE vector (49) that had been treated with thesame two enzymes. The DNA of the resultant plasmid p181AtENT3was used to transform fui1 cells. The uptake of [3H]uridine (AmershamBiosciences) by AtENT3 expressing fui1 cells in the absence or presenceof inhibitors (dilazep, dipyridamole, NBMPR, CCCP, or DNP) wasexamined as described above.

RESULTS

cDNA Cloning and Comparative Analysis of the DeducedAmino Acid Sequences of Arabidopsis ENTs—Using sequenceinformation generated from the AGI project, PCR primers(Table I) were synthesized for amplifying the cDNAs of thecomplete coding regions of seven potential ENTs (AtENTs 2–8)by RT-PCR. To maximize the chance of identifying the cDNAs,total RNA samples were prepared from suspension cells,leaves, stems, roots, flowers, or immature siliques. During theamplification experiments, it was common to find the cDNA forAtENTs 2, 3, 4, 6, 7, or 8. However, a cDNA for AtENT5 was notobtained in repeated trials. Further attempts involving the useof alternative PCR primers, Taq polymerases, and/or cyclingconditions did not lead to the identification of the cDNA specificfor AtENT5 either (data not shown). Sequencing the obtainedcDNAs revealed that, for AtENTs 2, 3, and 4, the coding regionsequences derived from cDNAs agreed with those predicted byAGI. In AtENTs 6, 7, and 8, the coding region sequences de-duced from cDNAs differed from those predicted by AGI. Acomparison of the cDNA sequences, the predicted coding regionsequences, and the genomic sequences of AtENTs 6–8 showedthat the disagreement was caused by imprecise prediction ofthe intron and exon boundaries during the annotation processof the AGI project (data not shown).

The amino acid sequences of AtENTs 2, 3, 4, 6, 7, and 8 werededuced from their respective cDNAs and compared with thoseof AtENT1 and ENTs from other sources (Table II). The iden-tities among the amino acid sequences of the seven AtENTsranged from 27.0% (between AtENT1 and AtENT4) to 91.1%(between AtENT3 and AtENT6) (Table II). AtENT1 andAtENT8 were more similar to each other (47.6% identical in

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amino acid sequences) than either to AtENTs 2, 3, 4, 6, and 7(Table II). AtENTs 2, 3, 4, 6, and 7 were more closely interre-lated (at least 50% identical in pairwise comparisons of aminoacid sequences) than any of them to AtENTs 1 and 8 (Table II).Amino sequence identities among AtENTs and ENTs fromother sources (mammals, Caenorhabditis elegans, S. cerevisiae,parasites) varied from 11.0% (between AtENT2 and Leishma-nia donovani NT2) to 26.5% (between AtENT8 and rat ENT1)(Table II). In phylogenetic analysis, the seven AtENTs formed

an independent cluster, which contained two subgroups, onecomposed of AtENTs 1 and 8 and the other of AtENTs 2, 3, 4,6, and 7 (Fig. 1). Because of the existence of significant levels ofidentities, the amino acid sequences of the seven AtENTs couldbe aligned with those of hENT1 and hENT2 (Fig. 2). Aided bythe alignment and the computer software HMMTOP, 11 puta-tive transmembrane domains were predicted for AtENTs 2, 3,4, 6, 7, and 8, respectively (Fig. 2). In the amino acid positioncorresponding to the residue 33 of hENTs 1 and 2, an Ile was

TABLE IPCR primers used in this study

For normalizing cDNA contents of reverse transcriptions, the primer set, TuF and TuR, was used. Their sequences were 5�-GAGGGACTATG-GCCGTTTAGG-3� and 5�-CACTTCACCCGACCATTCAATGG-3�. The underlined nucleotides constitute BamHI (GGATCC), EcoRI (GAATTC),NotI (GCGGCCGC) or SalI (GTCGAC) restriction sites.

AGI code AtENT Primer name Primer sequence Usagea

At1g70330 1 1F 5�-CTAAACGGATCCAAATGACCACCAC-3� B1R 5�-CGAAGCTTAAAGAATTCAAC-3�

At3g09990 2 2F 5�-GTCTGGATCCATGGATACTAGCATTCTA-3� A, B2R 5�-CAACGGATCCACCCAATCTTTACCAACTA-3�

At4g05120 3 3F 5�-TTGGTCGACATGGCGGATAGATATGAG-3� A3R 5�-GATTGTCGACGCAAAGGCATTCTTCTTAC-3�3F1 5�-GCGGATAGATATGAGAACCAACC-3� B3R1 5�-CCCTCCTAACAGAAATATCACCAGT-3�3RS 5�-GATGTCGACTCAAAAGGCATTCTTC-3� C3F2 5�-GCAGAATTCATGGCGGATAGATATGAG-3� D3R2 5�-GGTGCGGCCGCTCAAAAGGCATTCTTCTTA-3�

At4g05130 4 4F 5�-TTGTCGACATGGCGGATGGATACGAG-3� A4R 5�-ATGGTCGACCAGAAAGCATATTTCTTGC-3�4F1 5�-CATACCGCGAATCGAAAATCA-3� B4R1 5�-AGCATATTTCTTGCCAATAAGCCA-3�

At4g05140b 5 5F 5�-GAGTCGACATGGTGGCTAGGTTCGAG-3� A5R 5�-ATGGTCGACCAGAAGGCATTTTTCTTAC-3�

At4g05110 6 6Fc 5�-ATTGTCGACATGGCGGATATATACG-3� A6F1 5�-GCGGATATATACGAGCACCAAGT-3� B6R1 5�-GAGATAACGTGACAAAACCGCA-3�

At1g61630 7 7F 5�-ATGGATCCATGACTAATCCAGAGGA-3� A, B7R 5�-ATGGATCCTCAAACGAATCGTTGCCA-3�

At1g02630 8 8F 5�-GGAGGATCCATGGTTGATGAGAAAGTG-3� A, B8R 5�-TATGGATCCCTGATGAGCCAGAGCCAACC-3�

a A, for cDNA amplification; B, for transcript detection by semiquantitative RT-PCR; C, for constructing pYAtENT3 (in conjunction with primer3F); D, for constructing p181AtENT3.

b The primer combination (5F, 5R) was one of the seven primer sets that were designed and used in attempting to amplify the cDNA, or detectingthe transcripts, of At4g05140.

c Because of a high level of nucleotide sequence identity between the coding regions of At4g05110 and At4g05120, primers 6F and 3R could beused for amplifying the cDNA of At4g05110.

TABLE IIPercentages of amino acid sequence identities among AtENTs and functionally characterized ENTs from other sources

AtENTa GenBankTM

no.Identity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 AtENT1 AAF26446 29.5 28.7 27.0 28.2 27.8 47.6 24.3 25.7 24.5 24.3 23.8 24.1 25.7 22.0 17.5 17.3 15.4 15.2 15.2 16.8 15.42 AtENT2 AAL25095 57.8 52.8 55.6 51.8 27.5 16.3 16.5 14.4 16.3 14.9 15.6 17.5 16.5 14.1 14.1 11.0 15.3 17.0 16.1 12.73 AtENT3 AAL25096 82.5 91.1 62.8 29.8 17.5 17.9 16.5 18.9 17.0 17.0 19.6 17.2 16.0 16.3 14.4 15.8 15.8 15.6 11.24 AtENT4 AAL25097 80.4 59.7 27.5 20.1 18.9 17.5 17.9 17.5 17.0 18.4 17.7 17.2 17.5 12.2 15.3 15.8 13.4 14.85 AtENT6 AAL25098 60.9 29.3 17.9 18.2 17.2 20.3 17.7 17.5 19.4 17.2 14.1 14.1 12.4 15.3 15.8 15.8 15.66 AtENT7 AAL25094 29.6 18.2 19.9 19.4 18.5 20.4 20.6 18.2 18.0 16.3 16.8 15.1 15.1 15.3 16.5 16.57 AtENT8 AAO31974 26.2 24.9 26.5 24.7 25.2 25.2 27.2 17.5 18.8 18.5 20.6 17.5 18.5 21.3 11.88 hENT1 AAC51103 46.7 78.3 47.1 80.0 79.4 46.1 15.8 20.6 20.4 14.0 15.9 13.4 16.9 16.49 hENT2 AAC39526 46.5 87.9 45.6 46.1 88.6 18.4 18.4 16.9 18.0 16.1 14.3 17.5 14.7

10 rENT1 AAB88049 46.1 90.2 89.7 46.3 14.7 17.9 17.7 14.2 16.4 10.5 15.3 14.911 rENT2 AAB88050 46.1 46.3 94.5 18.9 18.0 17.8 15.4 16.8 12.3 20.0 18.012 mENT1.1 AAF76429 99.8 46.1 15.2 21.7 21.5 15.7 15.9 11.3 17.4 13.713 mENT1.2 AAF76430 46.3 14.8 20.1 19.9 14.8 16.8 12.2 17.7 14.614 mENT2 AAF76431 15.8 18.2 18.0 15.8 16.6 13.8 18.2 17.815 FUN26 AAC04935 10.8 10.8 16.6 15.2 13.2 17.3 17.516 LdNT1.1 AAC32597 96.8 29.7 11.6 27.2 29.2 18.017 LdNT1.2 AAC32315 29.5 11.6 27.2 29.4 18.018 LdNT2 AAF74264 14.2 41.3 43.6 13.919 PfENT1 AAG09713 11.8 16.1 16.420 TbAT1 AAD45278 57.7 14.521 TbNT2 AAF04490 14.122 TgAT AAF03427

a rENT, rat ENT; mENT, mouse ENT; LdNT, L. donovani nucleoside transporter; PfENT, Plasmodium falciparum ENT; TbAT, Trypanosomabrucei brucei adenosine transporter; TbNT, T. brucei brucei nucleoside transporter; TgAT, Toxoplasma gondii adenosine transporter; FUN,function unknown now.

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found for AtENTs 1 and 8 and a Leu for AtENTs 2, 3, 4, 6, and7 (Fig. 2, indicated by an arrow). The glycine residue, which islocated at the 179-position of hENT1 and important for theinteraction of hENT1 with inhibitors and substrates, was con-served at the corresponding positions of hENT2 and all sevenAtENTs (Fig. 2, indicated by inverted filled triangle). However,the glycine residue at the 154-position of hENT1 required forNBMPR binding was not conserved in either hENT2 or theseven AtENTs (Fig. 2, indicated by inverted open triangle).

Expression Patterns of AtENTs—The existence of multipleENTs in A. thaliana raises the question of whether they are allinvolved in the function of the salvage pathway of nucleotidesynthesis or if their expression possesses organ specificity. Toobtain evidence on the function of AtENTs in the salvage path-way of nucleotide synthesis, we attempted to inhibit the denovo pathway of nucleotide synthesis in Arabidopsis suspen-sion cells by nitrogen deprivation or application of fluorouraciland methotrexate, followed by investigating changes in thetranscript levels of the different AtENTs using semiquantita-tive RT-PCR. Both treatments led to clear increases in thetranscript levels of AtENTs 1, 3, 4, 6, and 8 (Fig. 3, A and B).Resumption of nitrogen supply or withdrawal of fluorouraciland methotrexate from the growth medium decreased the tran-script levels of AtENTs 1, 3, 4, 6, and 8 to those in the controlcell samples (data not shown). During these experiments, wewere unable to detect the transcripts of AtENTs 2 and 7 ineither control or treated suspension cell samples. Nevertheless,the results shown in Fig. 3, A and B, demonstrated that thetranscription of multiple AtENTs was elevated in response tothe inhibition of the de novo pathway of nucleotide synthesis inArabidopsis suspension cells.

To address the second question, the transcript levels of theseven AtENTs in five Arabidopsis organs were compared insemiquantitative RT-PCR experiments. The transcripts ofAtENT1 accumulated abundantly in all five organs (Fig. 3C).In contrast, the transcription of the remaining six AtENTsshowed varying degrees of organ specificities (Fig. 3C). The

transcripts of AtENT3 were undetectable in stem, whereasthose of AtENT4 were not found in either root or silique (Fig.3C). The transcripts of AtENT7 were not detected in root, stem,or silique although it was highly expressed in both leaf andflower (Fig. 3C). The seven AtENTs were all expressed in leafand flower (Fig. 3C). In contrast, fewer AtENTs were expressedto high levels in root (AtENTs 1 and 3), stem (AtENTs 1, 4, and8), or silique (AtENTs 1, 3, 6, and 8) (Fig. 3C).

Transport Activities of AtENT3—Before studying the trans-port activities of AtENT3, an AtENT3:EGFP fusion cistron wasexpressed in both insect and yeast cells. Confocal microscopy ofGFP fluorescence showed that the AtENT3:EGFP fusion pro-tein was localized specifically to the plasma membrane in ei-ther yeast or insect cells (data not shown). An AtENT3:GFPfusion cistron was also constructed and expressed in plantcells. In the control cell expressing the GFP cistron (Fig. 4A),the GFP fluorescence was distributed throughout the entirecell. In contrast, in the cell expressing the AtENT3:GFP fusioncistron (Fig. 4B), the GFP fluorescence was localized to theperiphery of the cell. A clear association of the AtENT3:GFPfusion protein with the plasma membrane (Fig. 4C, indicatedby arrows) but not the cell wall (Fig. 4C, indicated by arrow-heads) was found when the expressing cells were subject toosmotic shock. The same treatment did not change the distri-bution of GFP throughout the cytoplasm in the control exper-iment (Fig. 4D).

Following the strategies and methods described under “Ex-perimental Procedures,” five expression constructs (pY-AtENT1, pYAtENT1:GFP, pYAtENT3, and pYAtENT3:GFP)as well as the control vector pYES2 were introduced into thecells of the BMA64–1A ade2 strain of S. cerevisiae. After in-ducing the expression of the cloned sequences by galactose, theade2 cells harboring AtENT1 or AtENT3 coding sequencescould grow in the presence of 150 �M adenosine (Fig. 5 and datanot shown), indicating that adenine dependence of the ade2cells was rescued by adenosine transport activities of the pro-teins expressed from the cloned sequences in the four expres-

FIG. 1. Phylogenetic relationshipsof seven AtENTs to mammalian,yeast, and parasite ENTs with knowntransport activities. The rootless treewas constructed using the neighboringjoining method (with P distance and pair-wise deletion options). Bootstrap valuesare percentages of 500 replications. TheGenBankTM accession numbers for theproteins under comparison are shown inTable II.

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sion constructs. Using the ade2 cells expressing pYAtENT1,the main characteristics of adenosine transport mediated byAtENT1 were investigated. The Km and Vmax of adenosine

transport by AtENT1 were 3.6 �M and 134.7 pmol/mg protein/min, respectively. Adenosine transport by AtENT1, althoughresistant to dilazep and NBMPR, was abolished by CCCP.

FIG. 2. Multiple alignment of the deduced amino acid sequences of seven AtENTs with those of hENT1 and hENT2. The 11 putativetransmembrane helices are underlined. The arrow indicates amino acid residues in the seven AtENTs that correspond to amino acid residue 33of hENT1 and hENT2. The inverted open triangle marks the glycine residue at the 154-position of hENT1. The inverted filled triangle indicatesthe glycine residue at the 179-position of hENT1 that is conserved in hENT2 and all seven AtENTs.

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These results were comparable with those obtained by Mohl-mann et al. (13) on adenosine transport by AtENT1 using theoriginal W303 ade2 strain of S. cerevisiae.

Following the above experiments on AtENT1, the character-istics of nucleoside transport mediated by AtENT3 were inves-tigated in more detail. In initial uptake experiments, we foundthat BMA64–1A ade2 cell cultures expressing AtENT3 orAtENT3:GFP can both transport [3H]adenosine, but the uptakerate by AtENT3:GFP-expressing cells was generally 10% lower

than that by AtENT3-expressing cells. Therefore, yeast cellsexpressing AtENT3 rather than its GFP fusion protein wereused for subsequent experiments. In initial time course exper-iments with [3H]adenosine or [3H]uridine, the uptake of bothradiolabels was approximately linear for the first 5 min. Con-sequently, uptake periods of 2 min were employed in subse-quent experiments in order to estimate initial rates of sub-strate transport. The Km and Vmax of adenosine transport byAtENT3 expressed in the BMA64–1A ade2 strain were foundto be 2.9 �M and 269.9 pmol/mg protein/min, respectively (Fig.

FIG. 3. Transcript levels of AtENTs in Arabidopsis suspension cell samples (A and B) or in the different organs of Arabidopsisplants grown under normal physiological conditions (C). A, increased transcription of AtENTs 1, 3, 4, 6, and 8 induced by nitrogendeprivation. Samples were taken from the nitrogen-deprived (N�) and the control (nitrogen-sufficient, N�) suspension cell cultures at the indicatedtime points (days). Total RNA was extracted from the harvested cell samples and was converted to cDNA by reverse transcription. The cDNAcontents in the different reverse transcription mixtures were normalized by amplifying tubulin transcripts (bottom panel). AtENT transcript levelswere then evaluated using semiquantitative PCR. B, enhanced transcription of AtENTs 1, 3, 4, 6, and 8 augmented by drug (5-fluorouracil plusmethotrexate; 5-FU � MTX) treatment. The strategies for sample collection and evaluation of AtENT transcript levels were similar to those in A(except that nitrogen-sufficient medium was used for both control and drug-treated cell cultures). C, relative transcript levels of AtENTs 1, 2, 3,4, 6, 7, and 8 in the five organs (root, stem, leaf, flower, and silique) of Arabidopsis plants evaluated using semiquantitative PCR. The cDNAcontents of all transcription reactions were normalized by amplifying tubulin transcripts (bottom panel) prior to semiquantitative amplificationsof AtENT transcripts.

FIG. 4. Localization of AtENT3:GFP fusion protein to theplasma membrane in onion epidermal cells. Onion epidermal cellswere transiently transformed with the GFP (A and D) or AtENT3:GFP(B and C) expression cassettes using particle bombardment (46). Thesubcellular distribution of the ectopically expressed proteins was re-vealed by examining the location of GFP fluorescence using confocalmicroscopy. In C and D, the bombarded epidermal strips were subject toosmotic shock (by 0.8 M mannitol) for 15 min before being examined forGFP fluorescence. The arrows indicate the association of AtENT3:GFPwith the plasma membrane (C). The filled arrowheads mark the cellwall (C). Bars, 100 �m.

FIG. 5. Adenosine utilization mediated by recombinantAtENT3 or AtENT3:EGFP fusion protein in the BMA64–1A ade2strain of S. cerevisiae. A, plain recipient cells (1), cells transformedwith the control vector pYES2 (2), cells containing the expression vectorpYAtENT3 (3), and cells harboring the expression vector pYAtENT3:EGFP (4) were cultured on synthetic medium lacking uracil. B, thesame four cell lines were inoculated on synthetic medium lacking uraciland adenine but containing 150 �M adenosine. Only the cells containingthe expression vector pYAtENT3 (3) or pYAtENT3:EGFP (4) couldgrow, indicating that the ectopically expressed AtENT3 or AtENT3:EGFP fusion proteins can mediate the uptake of exogenously suppliedadenosine.

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6A). The corresponding values of uridine transport by AtENT3expressed in the fui1 strain were 3.2 �M and 232.5 pmol/mgprotein/min, respectively (Fig. 6B). CCCP reduced [3H]ade-nosine transport of AtENT3 by about 12% (Fig. 6C, Table III).DNP inhibited [3H]adenosine transport of AtENT3 by �38%(Table III). A similar effect of CCCP and DNP on [3H]uridinetransport of AtENT3 was also found (Fig. 6D, Table III). Vas-odilator drugs partly inhibited [3H]adenosine and [3H]uridinetransport by AtENT3 (Table III). However, AtENT3 transportof the two nucleosides was strongly resistant to NBMPR (TableIII). Employing [3H]adenosine, the substrate specificity ofAtENT3 was further investigated. In competition experiments,AtENT3 transport of [3H]adenosine was most significantly andconsistently reduced by both purine and pyrimidine nucleo-sides and 2�-deoxynucleosides (Table IV). In addition, certainnucleobases (cytosine and uracil) and nucleotides (e.g. ADP and

ATP) also inhibited [3H]adenosine uptake albeit at lower effi-ciencies (Table IV).

DISCUSSION

As a model species, A. thaliana has frequently been used instudying fundamental questions in higher plants. ENTs existin a wide variety of organisms, including higher plants (5, 6, 33,50, 51). However, their roles in plant biology remain to beinvestigated in a systematic manner. The complete sequencingof the genome of A. thaliana makes possible to investigate andcompare the functions of ENTs in a higher plant species from awhole genome level. But prior to the results reported in thispaper, only a limited amount of information had been obtainedon one (AtENT1) of the eight predicted ENTs in A. thaliana (13,33). In this study, the cDNAs for AtENTs 2, 3, 4, 6, 7, and 8were identified and sequenced. Despite repeated efforts, we

FIG. 6. Characters of [3H]adenosine and [3H]uridine transport by recombinant AtENT3. [3H]Adenosine transport was carried out usingthe BMA64–1A ade2 cells expressing AtENT3, whereas [3H]uridine transport was conducted using the AtENT3 expressing fui1 cells. A and B,kinetics of adenosine or uridine transport into intact yeast cells. Transport was allowed to proceed for 2 min in the indicated concentrations ofsubstrates. Uptake rates and S.E. values were calculated using the data (after subtracting the uptake rates of control cells) from three independentassays. The Km and Vmax values were derived from nonlinear fit to the uptake plot. Under such conditions, the Km and Vmax of adenosine transportby AtENT3 were 2.9 �M and 269.94 pmol/mg of protein/min, respectively, whereas the corresponding values of uridine transport by AtENT3 were3.2 �M and 232.5 pmol/mg of protein/min, respectively. The inserted Eadie-Hofstee plots are for illustration purposes. C and D, effect of theprotonophore CCCP on adenosine or uridine transport by recombinant AtENT3 in time course experiments. Yeast cells expressing AtENT3 weresuspended in a transport medium containing 1.8 �M [3H]adenosine or [3H]uridine (filled circles) or in the transport medium containing an identicalconcentration of the radioactive nucleoside plus 5 �M CCCP (open circles). Uptake rates and S.E. values were calculated using the data from threeindependent assays. As a control, [3H]adenosine or [3H]uridine uptake (in the absence of CCCP) by yeast cells harboring the empty vector (pYES2for adenosine uptake, p181AINE for uridine uptake) was also determined (filled triangles).

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failed to identify a cDNA for AtENT5. The failure may becaused by one or more of the following possibilities. The geneencoding AtENT5 may not be transcribed, its transcript levelmay be too low to be detected by RT-PCR, or its expression maybe highly regulated both spatially and temporally.

Amino acid sequence comparisons indicated that the sevenAtENTs are more closely related to each other than any of themare to ENTs from other sources. It is therefore understandablethat the seven AtENTs formed an independent cluster in phy-logenetic investigation. The same analysis also showed that theseven AtENTs could be divided into two subgroups (one con-taining AtENTs 1 and 8, the other containing AtENTs 2, 3, 4,6, and 7), indicating that there may be genetic and/or func-tional differentiation among ENTs from a single plant species.AtENTs and their counterparts from mammals, C. elegans,S. cerevisiae, and parasites all have 11 putative TMs, suggest-

ing that the conservation of 11 transmembrane TMs may beessential for all ENTs to transport their substrates and/orinteract with cellular membrane systems. It is interesting tonote that the glycine residues located at the 179-position ofhENT1 was conserved at the corresponding positions of hENT2and all seven AtENTs. Further experiments will be carried outto assess the importance of this residue in maintaining thestructure and transport function of AtENTs. In all sevenAtENTs, the position corresponding to the glycine residue atthe 154-position of hENT1 was occupied by Asp. In previousmutagenesis experiments, it has been found that a conserva-tive substitution of this Gly residue with Ser resulted in amutated hENT1 protein with reduced sensitivity to NBMPR(25). Because the size and charge properties of Asp are verydifferent from those of Gly or Ser, it will be interesting to testwhether the presence of Asp at this position is associated withthe strong resistance of AtENT3 (or AtENT1) to NBMPR.Finally, in all seven AtENTs, the position corresponding to resi-due 33 of hENT1 was occupied by one of two aliphatic aminoacids (Ile in AtENTs 1 and 8, Leu in AtENTs 2, 3, 4, 6, and 7).Because it has been shown that the mutation of residue 33 fromMet to Ile in hENT1 caused a significant reduction in dilazep anddipyridamole sensitivity (26), it will be important to examinewhether the presence of aliphatic amino acids in the positionscorresponding to the residue 33 of hENT1 is associated with theresistance of AtENT1 to dilazep and dipyridamole observed pre-viously (13) or with the relative insensitivity of AtENT3 todilazep demonstrated in the current study (see below).

In Arabidopsis suspension cells, the transcription of AtENTs1, 3, 4, 6, and 8 was clearly increased by nitrogen deprivation orthe application of fluorouracil and methotrexate. Nitrogen dep-rivation can have many consequences on the metabolism andgrowth of plant suspension cells. One such consequence may bethe inhibition of the de novo pathway of nucleotide synthesis,because the execution of the pathway needs low molecularweight nitrogenous precursors, the synthesis of which relies onthe absorption of nitrogen nutrient from the culture medium. Itis possible that nitrogen deprivation reduced the efficiency ofthe de novo pathway of nucleotide synthesis and lowered thenucleotide pool of the suspension cells. This led to an increasein the activity of the salvage pathway of nucleotide synthesis,which required enhanced transcription (expression) of multipleAtENTs. Thus, the results of our nitrogen deprivation experi-ments provided useful evidence on the function of multipleAtENTs in the salvage pathway of nucleotide synthesis. An-other important piece of evidence on the function of AtENTs inthe salvage pathway of nucleotide synthesis came from theexperiments using fluorouracil and methotrexate. Fluorouraciland methotrexate inhibit thymidylate synthetase and dihydro-folate reductase, respectively (52). Because both enzymes arerequired for dTMP synthesis through the de novo pathway,their inhibition may result in depletion of the dTMP pool in thesuspension cells. To compensate for the reduction of cellulardTMP pool, the synthesis of dTMP via the salvage pathwaymay be augmented, which would require increased transcrip-tion (expression) of multiple AtENTs. Because the transcrip-tion of AtENTs 2 and 7 was undetectable in the suspensioncells, their potential role in the salvage pathway of nucleotidesynthesis could not be investigated in our nitrogen deprivationor drug treatment experiments using suspension cells. BecauseAtENTs 2 and 7 were transcribed in leaf and flower in planta,their potential role in nucleoside transport (salvage) will beinvestigated in future using intact plants.

Under normal growth conditions, AtENT1 was expressedconstitutively and abundantly. In contrast, the transcription ofthe remaining six AtENTs showed varying degrees of organ

TABLE IIIInhibition of AtENT3-mediated [3H]adenosine or

[3H]uridine transport by inhibitorsTransport was performed at a substrate concentration of 1.8 �M for 8

min. [3H]Adenosine transport was carried out using the BMA64–1Aade2 cells expressing AtENT3, whereas [3H]uridine transport was con-ducted using the AtENT3-expressing fui1 cells. The concentration ofthe inhibitor applied is shown in parentheses. Means � S.E. werecalculated based on results of three independent assays.

Additive [3H]Adenosinetransport

[3H]Uridinetransport

% of control % of control

None 100 100Protonophore

CCCP (5 �M) 88.36 � 0.81 89.54 � 1.03DNP (1 mM) 62.23 � 1.60 67.39 � 2.56Dilazep (20 nM) 69.90 � 0.97 71.58 � 1.21

Vasodilator drugDilazep (20 �M) 38.67 � 3.80 32.18 � 4.56Dipyridamole (20 nM) 73.56 � 2.31 75.41 � 2.34Dipyridamole (20 �M) 42.86 � 4.21 29.97 � 6.07

Nucleoside analogNBMPR (20 nM) 97.51 � 0.47 95.39 � 0.66NBMPR (20 �M) 63.00 � 1.71 67.18 � 2.13

TABLE IVInhibition of AtENT3-mediated [3H]adenosine transport in yeast cells

by nucleosides, nucleobases, and nucleotides[3H]Adenosine transport was performed at a substrate concentration

of 1.8 �M for 8 min. The concentrations of various additives were 18 �M.Means � S.E. were calculated based on results of three independentassays.

Additive [3H]Adenosine transport

% of control

None 100Nucleosides

Adenosine 30.94 � 4.09Cytidine 40.78 � 3.01Guanosine 46.39 � 2.83Uridine 35.81 � 3.28Thymidine 25.94 � 4.562�-Deoxyadenosine 29.09 � 3.752�-Deoxycytidine 37.24 � 2.972�-Deoxyguanosine 29.01 � 3.872�-Deoxythymidine 15.01 � 4.49

NucleobasesAdenine 94.14 � 1.11Cytosine 57.34 � 2.68Guanine 99.29 � 0.29Uracil 57.15 � 2.62Hypoxanthine 99.84 � 0.14

NucleotidesADP 52.21 � 2.65ATP 57.46 � 3.20CTP 75.88 � 1.20GTP 86.38 � 0.88UTP 96.46 � 0.72

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specificities. All AtENTs were expressed in leaf and flower,suggesting that nucleoside salvage (and the de novo pathway ofnucleotide synthesis) may be an integral part of the physiologyof the two organs. Past studies have shown that in higherplants nucleotides can be synthesized via both de novo andsalvage pathways in leaf tissues and that there are nucleotides(such as ATP) and derivatives (e.g. ADP, AMP, adenosine, etc.)in the intercellular space of leaf cells (53–55). Furthermore,when leaves undergo senescence, nucleic acids in leaf cells aredegraded by various nucleases and phosphatases to yieldnucleosides, nucleobases, and phosphate for reuse in thegrowth of newer organs (56). It is therefore probable that ahigher level of nucleoside transport activities is maintained inthe leaf cells for transport and salvage of nucleosides. Flowerdevelopment in higher plants involves the formation of variousfloral meristems through mitosis and the development of ga-metic cells via meiosis. Both processes require the replication ofgenomic DNA, for which a sufficient supply of nucleotide pre-cursors is essential. It is possible that, in flowers, the twopathways of nucleotide synthesis are both operated in order tomaintain an adequate supply of nucleotide precursors. If the denovo pathway is inhibited due to nutrient deficiency (by lack ofsoil nitrate, etc.) or inhibition of photosynthesis (by environ-mental stresses such as drought, etc.), the salvage pathwaywould still ensure some supply of the nucleotides required forthe formation of floral meristematic cells and gametic cells. Theexpression of all seven AtENTs in the flower may thus be areflection of the importance of the salvage pathway in thedevelopment of flower and associated gametic cells. Evidencefor an essential role of the salvage pathway of nucleotide syn-thesis in flower comes also from the finding that mutation of anenzyme required for the salvage pathway results in defectivepollen cells and male sterility in A. thaliana (57).

Compared with the leaf and flower, fewer AtENTs wereexpressed in the root, stem, and silique. AtENTs 1, 3, 6, and 8were strongly transcribed in the silique. This indicates thatnucleoside transport activities (and the salvage pathway ofnucleotide synthesis) may also be important in the develop-ment of the maternal (pod and seed coat) and embryonic (cot-yledon and embryonic axis) tissues in silique. AtENTs 1, 4, and8 were highly transcribed in the stem. They may be involved inloading and unloading of nucleosides and analogs (such asnucleoside cytokinins; see below) in the vascular system forlong distance transport. Only two AtENTs (1 and 3) weretranscribed to high levels in root. This indicates that nucleosidetransport (salvage) in the root may not be as extensive as thatin the leaf, flower, silique, and stem. Nucleosides may be pro-duced from nucleic acid degradation in decaying plant or mi-crobial cells in the soil. The two AtENTs expressed in the rootmay have a role in the salvage of the nucleosides present in therhizosphere. The root-expressed AtENTs 1 and 3 may also beinvolved in the transport of the phytohormone cytokinins. Cy-tokinins, which are adenine analogs, are synthesized mainly inthe root apical meristem (58, 59). They are transported fromroot to shoot through the xylem in the form of nucleosidecytokinins (58–60). Because there is currently little informa-tion on how nucleoside cytokinins enter into and exit from thexylem, it would be important to examine whether AtENTsexpressed in root (or stem) may aid the transport of cytokininsthrough the vascular system of higher plants. Taken together,our results suggest that nucleoside transport (salvage) may beimportant in all parts of an Arabidopsis plant. However, theextent of nucleoside transport (salvage) may differ among dif-ferent organs, which may mainly be controlled by differentialexpression of AtENTs 2, 3, 4, 6, 7, and 8. Judging from patternsof organ specificities and transcript levels, AtENTs 1 and 3 may

be considered as the major ENTs whose activities are morewidespread in A. thaliana. The activities of AtENTs 4, 6, and 8are less prevalent, and those of AtENTs 2 and 7 are morerestricted to specific organs.

Previous investigation has shown that AtENT1 expressed inthe W303 ade2 strain of S. cerevisiae was a high affinity aden-osine transporter (13). In the present study, we confirmedprevious results on major characteristics of nucleoside trans-port mediated by AtENT1 using an alternative ade2 strain ofS. cerevisiae and constructs prepared using an alternativeyeast expression vector. For our investigation on the nucleosidetransport activities of AtENT3, we first demonstrated thatAtENT3 expressed from the cloned cDNA was targeted to theplasma membrane of plant, yeast, or insect cells. Subsequently,we found that AtENT3 expressed in yeast cells transportedadenosine (Km, 2.9 �M) and uridine (Km, 3.2 �M) with highaffinity. Furthermore, AtENT3 is also likely to be a transportwith broad substrate specificity on the basis of the data fromthe competition experiments (Table IV). We have recently ob-tained evidence on the transport of nucleotides (AMP, ADP,and ATP) by AtENT3 expressed in the BMA64–1A ade2 strainof S. cerevisiae.2

The transport processes mediated by AtENT3 and AtENT1share both similarities and differences. The similarities includeresistance to nanomolar concentration of NBMPR and trans-port of both nucleosides and 2�-deoxynucleosides. The majordifferences were in the following three aspects. First, protono-phores (CCCP and DNP) inhibited adenosine transport ofAtENT1 by almost 100% (13). In contrast, the inhibition ofAtENT3-mediated adenosine or uridine transport by CCCPand DNP was only around 10 and 35%, respectively (Table III).Second, adenosine transport of AtENT3 was partly inhibitedby nanomolar concentrations of dilazep and dipyridamole,whereas the two chemicals had no effect on adenosine trans-port by AtENT1 (13). Third, by expressing AtENT3 cDNA inthe fui1 strain of yeast, we obtained direct evidence on uridinetransport by AtENT3 (Table III), which is a common permeantof mammalian ENTs (61). However, AtENT1 was previouslydeduced not to transport uridine (13). Based on the abovecomparisons, we suggest that AtENT3 is an ei type ENT.

While investigating the effect of vasodilator drugs on nucle-oside transport of AtENT3, it was interesting to note that alarge increase in the concentration of dilazep or dipyridamolewas not followed by a more dramatic reduction in AtENT3’sactivity (Table III). If the two drugs were acting as competitiveinhibitors, the results obtained with of 20 nM would suggest aKi of �30 nM. With this calculation, the application of 20 �M

dilazep or dipyridamole would eliminate AtENT3 transportactivity. The present data, therefore, indicate that the twovasodilator drugs may act in a noncompetitive manner in in-hibiting nucleoside transport activity of AtENT3.

In conclusion, we have presented in this paper a more exten-sive study of the ENTs of A. thaliana predicted by genomesequencing. The cDNAs for seven of the eight predicted At-ENTs were cloned; their putative proteins could be subdividedinto two subgroups. The majority of the AtENTs may functionin the salvage pathway of nucleotide synthesis. Under normalgrowth conditions, there were at least two AtENTs highlytranscribed in each of five Arabidopsis organs, suggesting thatENT activities were important and regulated in all parts of anArabidopsis plant. In contrast to AtENT1 that was highlytranscribed in all five Arabidopsis organs and may function asa concentrative, proton-linked transporter, AtENT3, tran-scribed in most Arabidopsis organs, represented a typical ei

2 G. Li, K. Liu, S. A. Baldwin, and D. Wang, unpublished results.

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type ENT. The results and resources generated in this studyhave laid the foundation for more detailed comparative studieson mechanism of nucleoside transport, regulation, and biolog-ical role of AtENTs in the growth and development of A. thali-ana in the future.

Acknowledgments—We are grateful to Drs. Torsten Mohlmann andEkkehard Neuhaus of Universitat Kaiserslautern for providing theW303 yeast strain and help on functional expression of AtENT1 in yeastcells. In addition, we are grateful to Professor Carol Cass andZhang Jing for advice on methods of calculating the uptake rate ofradioactive nucleoside by yeast cells.

REFERENCES

1. Cass, C. E. (1995) in Drug Transport in Antimicrobial and Anticancer Chem-otherapy (Georgopapadakou, N. H., ed) pp. 403–451, Marcel Dekker, NewYork

2. Griffith, D. A., and Jarvis, S. M. (1996) Biochim. Biophys. Acta 1286, 153–1813. Baldwin, S. A., Mackey, J. R., Cass, C. E., and Young, J. D. (1999) Mol. Med.

Today 5, 216–2244. Young, J. D., Cheeseman, C. I., Mackey, J. R., Cass, C. E., and Baldwin, S. A.

(2001) in Gastrointestinal Transport: Molecular Physiology (Donowitz, M.,ed) Vol. 50, pp. 329–378, Academic Press, Inc., San Diego, CA

5. Hyde, R. J., Cass, C. E., Young, J. D., and Baldwin, S. A. (2001) Mol. Membr.Biol. 18, 53–63

6. Cabrita, M. A., Baldwin, S. A., Young, J. D., and Cass, C. E. (2002) Biochem.Cell Biol. 80, 623–638

7. Griffiths, M., Beaumont, N., Yao, S. Y., Sundaram, M., Boumah, C. E., Davies,A., Kwong, F. Y., Coe, I., Cass, C. E., Young, J. D., and Baldwin, S. A. (1997)Nat. Med. 3, 89–93

8. Vasudevan, G., Carter, N. S., Drew, M. E., Beverley, S. M., Sanchez, M. A.,Seyfang, A., Ullman, B., and Landfear, S. M. (1998) Proc. Natl. Acad. Sci.U. S. A. 95, 9873–9878

9. de Koning, H. P., Watson, C. J., and Jarvis, S. M. (1998) J. Biol. Chem. 273,9486–9494

10. Sanchez, M. A., Ullman, B., Landfear, S. M., and Carter, N. S. (1999) J. Biol.Chem. 274, 30244–30249

11. Maser, P., Sutterlin, C., Kralli, A., and Kaminsky, R. (1999) Science 285,242–244

12. Landfear, S. M. (2001) Biochem. Pharmacol. 62, 149–15513. Mohlmann, T., Mezher, Z., Schwerdtfeger, G., and Neuhaus, H. E. (2001)

FEBS Lett. 509, 370–37414. Huang, Q. Q., Yao, S. Y., Ritzel, M. W., Paterson, A. R., Cass, C. E., and Young,

J. D. (1994) J. Biol. Chem. 269, 17757–1776015. Parker, M. D., Hyde, R. J., Yao, S. Y., McRobert, L., Cass, C. E., Young, J. D.,

McConkey, G. A., and Baldwin, S. A. (2000) Biochem. J. 349, 67–7516. Sundaram, M., Yao, S. Y., Ingram, J. C., Berry, Z. A., Abidi, F., Cass, C. E.,

Baldwin, S. A., and Young, J. D. (2001) J. Biol. Chem. 276, 45270–4527517. Cass, C. E., Young, J. D., Baldwin, S. A., Cabrita, M. A., Graham, K. A.,

Griffiths, M., Jennings, L. L., Mackey, J. R., Ng, A. M., Ritzel, M. W.,Vickers, M. F., and Yao, S. Y. (1999) in Membrane Transporters as DrugTargets (Amidon, G. L., and Sadee, W., eds) Vol. 12, pp. 313–352, KluwerAcademic/Plenum Publishers, New York

18. Wohlhueter, R. M., Marz, R., Graff, J. C., and Plagemann, P. G. W. (1978)Methods Cell Biol. 20, 211–236

19. Belt, J. A. (1983) Mol. Pharmacol. 24, 479–48420. Belt, J. A. (1983) Biochem. Biophys. Res. Commun. 110, 417–42321. Belt, J. A., and Noel, L. D. (1985) Biochem. J. 232, 681–68822. Shi, M. M., and Young, J. D. (1986) Biochem. J. 240, 879–88323. Sundaram, M., Yao, S. Y., Ng, A. M., Griffiths, M., Cass, C. E., Baldwin, S. A.,

and Young, J. D. (1998) J. Biol. Chem. 273, 21519–2152524. Sundaram, M., Yao, S. Y., Ng, A. M., Cass, C. E., Baldwin, S. A., and Young,

J. D. (2001) Biochemistry 40, 8146–815125. Hyde, R. J., Abidi, F., Griffiths, M., Yao, S. Y., Sundaram, M., Phillips, S. E.,

Cass, C. E., Young, J. D., and Baldwin, S. A. (2000) Drug Dev. Res. 50, 3826. Visser, F., Vickers, M. F., Ng, A. M., Baldwin, S. A., Young, J. D., and Cass,

C. E. (2002) J. Biol. Chem. 277, 395–40127. SenGupta, D. J., Lum, P. Y., Lai, Y., Shubochkina, E., Bakken, A. H.,

Schneider, G., and Unadkat, J. D. (2002) Biochemistry 41, 1512–151928. Barros, L. F., Yudilevich, D. L., Jarvis, S. M., Beaumont, N., Young, J. D., and

Baldwin, S. A. (1995) Pflugers Arch. 429, 394–39929. Jennings, L. L., Hao, C., Cabrita, M. A., Vickers, M. F., Baldwin, S. A., Young,

J. D., and Cass, C. E. (2001) Neuropharmacology 40, 722–73130. Kombrink, E., and Beevers, H. (1983) Plant Physiol. 73, 370–37631. Kamboj, R. K., and Jackson, J. F. (1985) Plant Physiol. 79, 801–80532. Kamboj, R. K., and Jackson, J. F. (1987) Plant Physiol. 84, 688–69133. Li, J., and Wang, D. (2000) Plant Sci. 157, 23–3234. Murray, A. W. (1971) Annu. Rev. Biochem. 40, 811–82635. Berens, R. L., Krug, E. C., and Marr, J. J. (1995) in Biochemistry and Molec-

ular Biology of Parasites (Muller, M., ed) pp. 89–117, Academic Press, Inc.,New York

36. Sherman, I. W. (1998) in Malaria: Parasite Biology, Pathogenesis, and Protec-tion (Sherman, I. W., ed) pp. 177–184, American Society for MicrobiologyPress, Washington, D. C.

37. Van’t Hof, J. (1968) J. Cell Biol. 37, 773–78038. Lin, B. L., and Raghavan, V. (1991) Am. J. Bot. 78, 740–74639. Lopez, M. E., Giordano, O. S., and Lopez, L. A. (2002) Protoplasma 219, 82–8840. Hervas, J. P., de la Flor, J., and Santa-Cruz, M. C. (2002) Biotech. Histochem.

77, 145–15241. Li, D., Zhu, H., Liu, K., Liu, X., Leggewie, G., Udvardi, M., and Wang, D. (2002)

J. Biol. Chem. 277, 27772–2778142. Nei, N., and Kumar, S. (2000) Molecular Evolution and Phylogenetics, Oxford

University Press, Oxford, United Kingdom43. Tusnady, G. E., and Simon, I. (1998) J. Mol. Biol. 283, 489–50644. Hu, Y., Bao, F., and Li, J. (2000) Plant J. 24, 693–70145. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,NY

46. Silva, N. F., and Goring, D. R. (2002) Plant Mol. Biol. 50, 667–68547. Vickers, M. F., Yao, S. Y., Baldwin, S. A., Young, J. D., and Cass, C. E. (2000)

J. Biol. Chem. 275, 25931–2593848. Vickers, M. F., Kumar, R., Visser, F., Zhang, J., Charania, J., Raborn, R. T.,

Baldwin, S. A., Young, J. D., and Cass, C. E. (2002) Biochem. Cell Biol. 80,639–644

49. Daram, P., Brunner, S., Persson, B. L., Amrhein, N., and Bucher M. (1998)Planta 206, 225–233

50. Coe, I. R., and Acimovic, Y. (2002) Mol. Biol. Evol. 19, 2199–221051. Newman, T., de Bruijn, F. J., Green, P., Keegstra, K., Kende, H., McIntosh, J.,

Ohlrogge, J., Raikhel, N., Somerville, S., Thomashow, M., Retzel, E., andSomerville, C. (1994) Plant Physiol. 106, 1241–1255

52. Campbell, M. K. (1999) in Biochemistry, 3rd Ed., pp. 670–671, Harcourt BraceCollege Publishers, Orlando, FL

53. Ross, C. W. (1981) in The Biochemistry of Plants (Conn, E. E., ed) Vol. 6, pp.169–205, Academic Press, Inc., New York

54. Nazario, G. M., and Lovatt, C. J. (1993) Plant Physiol. 103, 1203–121055. Thomas, C., Sun, Y., Naus, K., Lloyd, A., and Roux, S. (1999) Plant Physiol.

119, 543–55156. Buchanan, B. B., Gruissem, W., and Jones, R. L. (2000) Biochemistry and

Molecular Biology of Plants, pp. 861–862, American Society of Plant Phys-iologists, Berkeley, CA

57. Moffat, B., and Sommerville, C. (1988) Plant Physiol. 86, 1150–115458. Torrey, J. G. (1976) Annu. Rev. Plant Physiol. 27, 435–45959. Fosket, D. E. (1998) in Plant Physiology (Zeiger, E., ed) 2nd Ed., pp. 621–650,

Sinauer Associates, Inc., Publishers, Sunderland, MA60. Nooden, L. D., and Letham, D. S. (1993) Aust. J. Plant Physiol. 20, 639–65361. Cass, C. E., Young, J. D., and Baldwin, S. A. (1998) Biochem. Cell Biol. 76,

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Comparative Analysis of Arabidopsis ENTs35742

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Guangyong Li, Kunfan Liu, Stephen A. Baldwin and Daowen WangEXPRESSION PATTERN, AND ANALYSIS OF TRANSPORT ACTIVITIES

: cDNA CLONING,Arabidopsis thalianaEquilibrative Nucleoside Transporters of

doi: 10.1074/jbc.M304768200 originally published online June 16, 20032003, 278:35732-35742.J. Biol. Chem. 

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