Amalgamating tools of genetics, molecular biology, biochemistry, and cell biology, this proposal offers an interdisciplinary analysis of the nucleoside transporters of Leishmania donovani and Trypanosoma brucei. As protozoan parasites are auxotrophic for purines, nucleoside transporters provide an important, if not vital, nutritional function for the parasite. These membrane carriers also mediate the translocation of melarsoprol and pentamidine, two anti-trypanosomal drugs, as well as allopurinol riboside and formycin B, two anti-trypanosomatid agents, into the parasite. Two nucleoside transporters have been genetically and biochemically defined for L. donovani and T. brucei. We have cloned, sequenced, and partially characterized the genes, LdNT1 and LdNT2, encoding the two L. donovani nucleoside transporters employing a functional rescue strategy of mutant nucleoside transport-deficient parasites. The sequences of LdNT1 and LdNT2 enable the subsequent isolation of two T.brucei nucleoside transport cDNAs, TbNT1 and TbNT2. These reagents serve as the molecular cornerstone of the three specific aims in this proposal.
Specific Aim 1 will be to characterize the adenosine/pyrimidine transporter, LdNT1. We will verify whether LdNT1 is a proton transporter by expression of LdNT1 in Xenopus oocytes, determine the subcellular location of LdNT1 by immunocytochemistry, and ascertain the nature of the genetic lesion(s) in the adenosine/pyrimidine transport-deficient strain, TUBA5.
Specific Aim II involves the characterization of LdNT2 will be completed. Next, LdNT2 function will be characterized after expression in both inosine/guanosine transport-deficient FBD5 parasites and oocytes. Whether LdNT2 is a proton supporter, the location of the protein within the parasite, and the nature of the mutation(s) in FBD5 cells will also be evaluated. The final specific aim will be to characterize TbNT1 and TbNT2 substrate specificities and ligand affinities by heterologous expression of the corresponding cDNAs in L. donovani and oocytes, to determine whether either transporter recognizes melarsoprol or pentamidine, and to discover whether three truncated versions of TbNT1 and TbNT2 encode functional nucleoside transporters.
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