Abstract
Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.
Highlights
Rimidine nucleobases and nucleosides into eukaryotic cells [1]
A selection for rare mutants that led to a gain of function, namely a change in ligand specificity, was used to identify regions of the C. fasciculata inosine/ guanosine transporter Crithidia fasciculata nucleoside transporter 2 (CfNT2) that were required for ligand discrimination rather than ligand translocation and/or proper protein folding
Thirty positive clones were subjected to plasmid sequencing, and the majority carried a mutation of Gly86, which maps to predicted TM2, or of Asp159, which is in the predicted TM4 (Fig. 1)
Summary
Cell Culture and Other Reagents—Escherichia coli DH5␣ and TOP10 strains were used throughout (Invitrogen), and standard methods for recombinant DNA work were employed [19]. Nucleoside Uptake in Leishmania—Log phase parasites (ϳ1 ϫ 107 cells/ml) were washed three times in ice-cold PBS plus 10 mM glucose and resuspended in the same buffer at a density of 2– 4 ϫ 108/ml. Duplicate tubes containing 5 M [3H]inosine plus a 500 M concentration of a potential inhibitor and buffer-matched duplicate control tubes without inhibitor were assayed in parallel at a single time point within the linear range of uptake by the oil-stop method (10 s for ⌬ldnt1/⌬ldnt pALTneo-HA-CfNT2 and 60 s for ⌬ldnt1/⌬ldnt pALTneoHA-cfnt2-K155N). Cell density was adjusted to 3– 4 ϫ 108/ml, and uptake of 1 M [3H]inosine and 1 M [3H]inosine plus 1 M unlabeled inosine was measured in triplicate at a single time point within the linear range of uptake as described above. Bader atomic charge analysis [36, 37] was performed using B3LYP hybrid exchange correlation [38], triple-zeta double polarization atomic basis, and COSMO simulation of water
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
