EFFECTS OF INHIBITION OF N-LINKED GLYCOSYLATION BY TUNICAMYCIN ON NUCLEOSIDE TRANSPORT POLYPEPTIDES OF L1210 LEUKEMIA-CELLS

Membrane polypeptides (relative mass (M(r) 48 000 - 55 000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (M(r) 42 000 - 47 000) during sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating from (M(r) 41 000 - 48 000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by > 95% had little, if any, effect on either the affinity (K(d) values, 0.1-0.2 nM) or abundance (B(max) values, 200 000 - 220 000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37°C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.