Abstract
Permeases of the equilibrative nucleoside transporter family mediate the uptake of nucleosides and/or nucleobases in a diverse array of eukaryotes and transport a host of drugs used for treatment of cancer, heart disease, AIDS, and parasitic infections. To identify residues that play central roles in transport function, we have systematically substituted by site-directed mutagenesis all the charged residues located within predicted transmembrane domains of the Leishmania donovani nucleoside transporter 1.1, LdNT1.1, which transports adenosine and the pyrimidine nucleosides. Substitution of three of these ten residues by uncharged amino acids resulted in loss of >95% transport activity, and we hence designated them "key" residues. These amino acids were Glu94, Lys153, and Arg404 located in transmembrane domains 2, 4, and 9, respectively. In addition, previous studies on the related LdNT2 inosine/guanosine transporter identified the highly conserved Asp389 and Arg393 (equivalent to Asp374 and Arg378 in LdNT1.1) in transmembrane domain 8 as key residues. Among these residues, the mutants in Arg393 (LdNT2) and Arg404 were strongly impaired in trafficking to the plasma membrane, but the other mutants were expressed with high to moderate efficiency at the cell surface, indicating that their mutation impaired transport activity per se. A conservative K153R substitution exhibited a change in substrate specificity, acquiring the ability to transport inosine, a nucleoside that is not a substrate for the wild-type LdNT1.1 permease. These results imply that the Glu94, Lys153, and Asp374 residues may play central roles in the mechanism of substrate translocation in LdNT1.1.
Highlights
Membrane transporters are responsible for the uptake of essential nutrients, modulation of concentrations of physiologically relevant compounds, and active release of substances such as signaling molecules
Functional Characterization of Mutants within transmembrane domains (TMDs) of LdNT1.1—To identify residues likely to play central roles in transport activity, we systematically evaluated the function in substrate permeation of all charged residues of LdNT1.1 located within predicted transmembrane segments
We used TMpred software [16] to infer that LdNT1.1 encompasses 11 putative transmembrane helices, with cytoplasmic N-terminal and extracellular C terminus (Fig. 1). This predicted topology is supported by experimental data defining the topology of the human equilibrative nucleoside transporter hENT1 [14, 29] and likely all ENT family members
Summary
Materials, and Reagents—Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs, Inc., Roche Pharmaceuticals, or Invitrogen. Null mutant ⌬ldnt (in which both alleles of the clustered LdNT1.1 and LdNT1.2 locus were deleted by targeted gene replacement [46] followed by loss-ofheterozygosity [47] to generate a homozygous null mutant for the entire locus was cultured continuously in 50 g/ml hygromycin (Roche Pharmaceuticals) for which the selective marker hygromycin phosphotransferase used in the gene replacement strategy confers resistance, and 1 M tubercidin (Sigma) to avoid possible contamination with wild-type parasites. Cell lines generated by transfection of ⌬ldnt with pX63NeoRI constructs (described below) were selected and maintained in 100 g/ml G418 (Invitrogen) and 50 g/ml hygromycin. Clones were selected by PCR screening, and mutations were confirmed again by DNA sequencing before being transfected into the transport-defective ⌬ldnt or ⌬ldnt1/⌬ldnt L. donovani cell lines. The ORFs were subcloned into the BamHI site of the pXGGFPϩ2Ј vector [49], and all constructs were transfected into the ⌬ldnt parasites as described.
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