Cloning and in vitro expression of the cDNA encoding a putative nucleoside transporter from Arabidopsis thaliana.

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 AtENT1 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 AtENT1 had one homologue in the Arabidopsis genome. Northern blot analysis showed that AtENT1 might be constitutively expressed in most Arabidopsis organs and in plants at different developmental stages. Two AtENT1 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.