Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surfa ce nucleoside and nucleobase transporters to provide them with host purines . Leishmania donovani possess two closely related genes that encode high af finity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the na tural substrates. In this study, we have characterized a drug-resistant clo nal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wildtype cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not con tribute to nucleoside transport in this stage of the life cycle. In contras t, the TUBAS cells were compound heterozygotes at the LdNT1.1 locus contain ing two mutant alleles that encompassed distinct-point mutations, each of w hich impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C 337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only sli ghtly elevated adenosine Km values, the severe impairment in transport resu lted from drastically (approximate to 20-fold) reduced V-max values. Becaus e these transporters were correctly targeted to the plasma membrane, the re duction in V,,, apparently resulted from a defect in translocation. Strikin gly, G183 was essential for pyrimidine nucleoside but not adenosine transpo rt. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridin e uptake. These results suggest that TM 5 is likely to form part of the nuc leoside translocation pathway in LdNT1.1.