Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae -: Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q)

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of H-3-labelled nitrobenzylmercaptopurine ribonucleoside (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosyl purine; NBMPR) to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means +/- S.D.) of [H-3]NBMPR to intact yeast (K-d 1.2 +/- 0.2 nM; B-max 5.0 +/- 0.5 pmol/mg of protein) and membranes (Kd 0.7 +/- 0.2 nM; B-max 6.5 +/- 1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [H-3]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130 +/- 10 and 380 +/- 20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [H-3]NBMBR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The K-d for binding of NBMPR to hENT1/N48Q was 10.5 +/- 1.6 nM, indicating that the replacement of an Asn residue with Gin decreased the affinity of hENT1 for NBMPR, The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (k(off)) and a decreased rate of association (k(on)) of specifically bound [H-3]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14 +/- 0.02 and 0.36 +/- 0.05 min(-1) for k(off), and (1,2 +/- 0.1) x 10(8) and (0.40 +/- 0.04) x 10(8) M-1 min(-1) for k(on). These results indicated that the conservative conversion of an Asn residue into Gin at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.