Cloning of Leishmania Nucleoside Transporter Genes by Rescue of a Transport-Deficient Mutant

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 17; pp. 9873 - 9878
• Main Authors: Vasudevan, Gayatri, Carter, Nicola S., Drew, Mark E., Beverley, Stephen M., Sanchez, Marco A., Seyfang, Andreas, Ullman, Buddy, Landfear, Scott M.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 18.08.1998; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: Amino Acid Sequence; Amino acids; Animals; Biological Sciences; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell lines; Cloning; Cloning, Molecular; Cosmids; Drug Resistance - genetics; Equilibrative Nucleoside Transporter 1; Female; Gene Expression; Genes; Genes, Protozoan; Genetic vectors; Humans; Leishmania donovani - drug effects; Leishmania donovani - genetics; Leishmania donovani - metabolism; Membrane Proteins - genetics; Molecular Sequence Data; Mutation; Nucleoside Transport Proteins; Nucleosides; Oocytes - metabolism; Open reading frames; Parasites; Phenotype; Protozoa; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Purine Nucleosides - metabolism; Purines; Restriction Mapping; Sequence Homology, Amino Acid; Tubercidin - pharmacology; Xenopus
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.17.9873

All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine/pyrimidine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Two such genes, LdNT1.1 and LdNT1.2, have been sequenced and shown to encode deduced polypeptides with significant sequence identity to the human facilitative nucleoside transporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 proteins predicted 11 transmembrane domains. Transfection of the adenosine/pyrimidine nucleoside transport-deficient TUBA5 parasites with vectors containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ability of this mutant line to take up [3H]adenosine and [3H]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significantly increases their ability to take up [3H]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. These results establish genetically and biochemically that both LdNT1 genes encode functional adenosine/pyrimidine nucleoside transporters.