title: Point mutations within the LdNT2 nucleoside transporter gene from Leishmania donovani confer drug resistance and transport deficiency.
abstract: Leishmania donovani, a protozoan parasite, expresses an unusual inosine/guanosine-specific transporter, LdNT2, the gene for which was cloned by functional rescue of a drug-resistant, LdNT2-deficient (FBD5) strain. In this investigation, we have uncovered and characterized the mutations within the LdNT2 open reading frame that are the basis for the drug-resistance and transport-incompetent phenotype of the FBD5 line. The FBD5 cells were shown to be compound heterozygotes in which both mutant ldnt2 alleles harbor discrete point mutations, each of which impaired transport function and conferred resistance to formycin B, the drug to which the clonal FBD5 line was selected. One of the mutant ldnt2 alleles encoded an S189L alteration in predicted transmembrane domain 5, while the second allele accommodated a null mutation at codon 376, which truncated the transporter just prior to transmembrane domain 8. In addition to the null transport phenotype, very little S189L ldnt2 mutant transporter targeted to the surface of the parasite. The bulk of the truncated ldnt2 appeared to be sequestered internally, possibly within the endoplasmic reticulum, but some of the truncated transporter seemed to be cell surface exposed. The ability to dissect mutations within a viable parasite offers LdNT2 as an attractive model for implementing a thorough forward genetic dissection of transporter function in a eukaryotic cell.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239-3098, USA.
authors.forename: Jon
authors.initials: J
authors.lastname: Galazka
authors.forename: Nicola S
authors.initials: NS
authors.lastname: Carter
authors.forename: Sabih
authors.initials: S
authors.lastname: Bekhouche
authors.forename: Shirin
authors.initials: S
authors.lastname: Arastu-Kapur
authors.forename: Buddy
authors.initials: B
authors.lastname: Ullman
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D007893:Leishmania donovani / Q000235:genetics* / Q000378:metabolism; D033703:Nucleoside Transport Proteins / Q000737:chemistry / Q000235:genetics*; D017354:Point Mutation; D021381:Protein Transport / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; C411852:LdNT2 protein, Leishmania donovani; D033703:Nucleoside Transport Proteins; D015800:Protozoan Proteins