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Plant Science 157 2000 23 – 32 Cloning and in vitro expression of the cDNA encoding a putative nucleoside transporter from Arabidopsis thaliana Jian Li, Daowen Wang The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics, The Chinese Academy of Sciences, Beijing 100101 , PR China Received 8 February 2000; received in revised form 23 March 2000; accepted 24 March 2000 Abstract Nucleoside transporters are integral membrane proteins involved in the uptake or release of nucleosides. Their function constitutes an essential step of the salvage pathway of nucleotide synthesis. In order to study the function of these proteins in higher plants, we cloned the cDNA corresponding to the AtENT 1 gene that encodes a putative nucleoside transporter in Arabidopsis thaliana by RT-PCR. The amino acid sequence of the AtENT1 protein deduced from the cloned cDNA shared similarity to those of eukaryotic equilibrative nucleoside transporters. Structure prediction indicated that the deduced AtENT1 protein might possess eleven putative transmembrane domains. Southern hybridization revealed that AtENT 1 had one homologue in the Arabidopsis genome. Northern blot analysis showed that AtENT 1 might be constitutively expressed in most Arabidopsis organs and in plants at different developmental stages. Two AtENT 1 fusion genes, AtENT1-His-tag and GFP-AtENT1-His-tag, were expressed in insect cells. Confocal microscopy demonstrated that the GFP-AtENT1-His-tag fusion protein was targeted specifically to the plasma membrane of insect cells. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Nucleoside transporter; cDNA cloning; In vitro expression; Arabidopsis thaliana www.elsevier.comlocateplantsci

1. Introduction

Considerable progress has been made in recent years in biochemical and molecular characteriza- tion of nucleoside transport NT processes in eukaryotic organisms such as mammals and proto- zoan parasites [1 – 9]. To date, seven types of NT processes have been described for mammalian cells [1,2]. The nucleoside transporters required for the different NT processes comprise two functionally different and structurally unrelated families of transmembrane proteins [1,2]. The equilibrative nucleoside transporters ENTs facilitate the diffu- sion of nucleosides down their concentration gra- dients [1 – 5]. According to their sensitivity to inhibition by nitrobenzylmercaptopurine ribonu- cleoside NBMPR, the ENTs can be subdivided into ei and es types. Transport mediated by the ei transporters is insensitive to NBMPR inhibition whereas that mediated by the es transporters is sensitive to inhibition by nanomolar concentra- tions of NBMPR. Nucleoside transport mediated by the ei and es transporters is bidirectional. The concentrative nucleoside transporters CNTs are Na + -dependent symporters [1 – 5]. They transport nucleosides unidirectionally and against their con- centration gradients. Based on their selectivity, the CNTs can be further divided into five types: cit, cif, cib, csg and cs. Nucleoside transport processes mediated by the five types of CNTs have recently been reviewed [1 – 5]. While the ENTs are present in most mammalian cell types, the CNTs are found primarily in specialized epithelia [1 – 5]. Some cell types can simultaneously possess several kinds of nucleoside transport activities, indicating the presence of multiple types of nucleoside trans- porters. Equilibrative nucleoside transport pro- Corresponding author: Tel.: + 86-10-64889380; fax: + 86-10- 64873482. E-mail address : dwwangigtp.ac.cn D. Wang. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 6 1 - 2 cesses and ENTs have been studied for protozoan parasites such as Leishmania dono6ani, Try- panosoma brucei and Toxoplasma gondii [6 – 9]. These parasites lack the de novo purine biosynthe- sis pathway and, consequently, have to rely on nucleoside transporters to acquire purine nu- cleosides from their host cells [10]. Genes encoding eukaryotic ENTs have been cloned from protozoan parasites and mammals [6 – 9,11 – 14]. Amino acid sequence comparisons suggest structural conservation of this type of genes between mammals and protozoan parasites. Genes encoding CNTs have mainly been isolated from mammalian cells [15 – 19]. A high degree of structural conservation is also found among the CNT encoding genes. Nucleoside transport func- tion has been confirmed for all of the cloned genes through in vitro expression in Xenopus lae6is oocytes followed by nucleoside uptake tests. More recently, the Xenopus and the yeast expression systems have been employed successfully for studying amino acid residues involved in nu- cleoside or inhibitor NBMPR binding in human CNTs hCNTs or ENTs hENTs [20,21]. The combination of molecular genetic and biochemical approaches is yielding new information on nu- cleoside transporters at a rapid pace. A deeper understanding of these proteins in mammalian and protozoan biology will become available before long. In contrast to above progress, fewer investiga- tions have been carried out to characterize nu- cleoside transporters in higher plants. Physiological studies suggest that higher plants can salvage nucleosides and bases derived from nucleotide breakdown or from exogenous sources [22 – 25], indicating the existence and function of nucleoside transporters in plant cells. Plant nucleic acid sequences encoding potential polypeptides with homology to hENTs have recently been dis- covered in EST expressed sequence tag [26] and genomic sequencing projects of Arabidopsis thaliana. In the work described in this paper, we report our results on cloning and in vitro expres- sion of the cDNA encoding a putative nucleoside transporter from Arabidopsis thaliana. Our work presents molecular evidence for the expression of the AtENT 1 gene in the Arabidopsis plant and for the association of the AtENT1 protein with the plasma membrane of eukaryotic cells.

2. Materials and methods