Abstract

In this study, we describe the cloning and characterization of a proton-dependent, broadly selective nucleoside transporter fromCaenorhabditis elegans. Recently, we constructed a broadly selective nucleoside transporter which accepts both purine and pyrimidine nucleosides. Based on these studies, we hypothesized that CNTs with novel substrate selectivities exist in nature and that a CNT homolog in the C. elegans genomic database may function as a broadly selective nucleoside transporter. We cloned the cDNA for this transporter, termed CeCNT3 because of its broad selectivity, using polymerase chain reaction-based methods. CeCNT3 is predicted to have 575 amino acid residues (63.4 kDa) with 11 to 14 putative transmembrane domains and exhibits ∼30% identity to members of the mammalian CNT family. This transporter exhibits a novel substrate selectivity, transporting a wide range of purine and pyrimidine nucleosides (inosine, guanosine, adenosine, uridine, and thymidine) but not cytidine. The apparent K _m values for inosine and thymidine are 15.2 ± 5.3 μM and 11.0 ± 2.4 μM, respectively. Kinetic studies demonstrate that purine and pyrimidine nucleosides share a common recognition site in the transporter. In contrast to all known members of the mammalian CNT family, CeCNT3-mediated transport of nucleosides is proton-, but not sodium-, dependent. Mutation of tyrosine 332 in CeCNT3 decreased both the maximum uptake rate and apparent K _m of thymidine, suggesting that this residue is in the domain of nucleoside recognition and translocation. The broad nucleoside specificity of CeCNT3 may be explained by this and other residues that restrict purine and pyrimidine nucleoside uptake and that discriminate among pyrimidine nucleosides.