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J. Biol. Chem., Vol. 278, Issue 37, 35732-35742, September 12, 2003

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Equilibrative Nucleoside Transporters of Arabidopsis thaliana

cDNA CLONING, EXPRESSION PATTERN, AND ANALYSIS OF TRANSPORT ACTIVITIES^*

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

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

Equilibrative nucleoside transporters (ENTs) occur in diverse organisms. In the model plant Arabidopsis thaliana, eight potential ENTs (AtENTs) have been predicted by genome sequencing. We here report the cloning of the cDNAs for AtENTs 2, 3, 4, 6, 7, and 8. Conceptual translation of the cDNAs of AtENTs 2, 3, 4, 6, 7, and 8 yielded polypeptides possessing strong similarities to ENTs characterized previously. Eleven putative transmembrane domains were identified in each of the six AtENTs. In suspension cells, the transcription of AtENTs 1, 3, 4, 6, and 8 was increased by two treatments (nitrogen deprivation, application of 5-fluorouracil and methotrexate) that inhibited the de novo pathway of nucleotide synthesis, indicating that multiple members of the Arabidopsis ENT family may function in the salvage pathway of nucleotide synthesis. Except for AtENT1, the transcription of the remaining six AtENTs showed varying degrees of organ specificity. However, all seven AtENTs were expressed in the leaf and flower. In plant, insect, and yeast cells, ectopically expressed AtENT3 was targeted to the plasma membrane. AtENT3 expressed in yeast cells transported adenosine and uridine with high affinity. Furthermore, the activities of AtENT3 appear not to require a transmembrane proton gradient because protonophores did not abolish adenosine or uridine transport. In competition experiments, the transport of [³H]adenosine by AtENT3 was most significantly inhibited by a number of different purine and pyrimidine nucleosides and 2'-deoxynucleosides, although certain nucleobases and nucleotides were also found to have some inhibitory effect. This indicates that AtENT3 may possess broad substrate specificity. Adenosine and uridine transport by AtENT3, although partly sensitive to the vasodilator drugs dilazep and dipyridamole, was resistant to the nucleoside analogue nitrobenzylmercaptopurine ribonucleoside. We conclude that AtENT3 represents the first ei type ENT characterized from higher plants. The potential functions of ENTs in the biology of A. thaliana are discussed.

Received for publication, May 7, 2003

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

^* This work was supported by National Natural Science Foundation of China Grant 39770491 and Chinese Academy of Sciences Grant KSCX2-SW-304. The costs of publication of this article were defrayed in part by the payment of page charges. This 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. Tel.: 86-10-64889380; Fax: 86-10-64854467; E-mail: dwwang{at}genetics.ac.cn or daowenwang{at}hotmail.com.

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