Details

Autor(en) / Beteiligte

• Vasudevan, Gayatri
• Ullman, Buddy
• Landfear, Scott M.

Titel

Point Mutations in a Nucleoside Transporter Gene from Leishmania donovani Confer Drug Resistance and Alter Substrate Selectivity

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 2001-05, Vol.98 (11), p.6092-6097

Ort / Verlag

United States: National Academy of Sciences

Erscheinungsjahr

2001

Quelle

MEDLINE

Beschreibungen/Notizen

• Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drugresistant clonal 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 wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Kmvalues, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmaxvalues. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmaxapparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1