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)
Mf. Vickers et al., 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), BIOCHEM J, 339, 1999, pp. 21-32
We have produced recombinant human equilibrative nucleoside transporter (hE
NT1) in the yeast Saccharomyces cerevisiae and have compared the binding of
inhibitors of equilibrative nucleoside transport with the wild-type transp
orter and a N-glycosylation-defective mutant transporter. Equilibrium bindi
ng of H-3-labelled nitrobenzylmercaptopurine ribonucleoside (6-[(4-nitroben
zyl)thio]-9-beta-D-ribofuranosyl purine; NBMPR) to hENT1-producing yeast re
vealed a single class of high-affinity sites that were shown to be in membr
ane 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 proteolipos
omes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibit
ed 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 NBMP
R 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 o
f 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 d
ecreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of d
issociation (k(off)) and a decreased rate of association (k(on)) of specifi
cally bound [H-3]NBMPR because the values for hENT1-producing and hENT1/N48
Q-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 transp
orter for NBMPR, dilazep and dipyridamole.