Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

Abstract

Membrane polypeptides (relative mass (Mr) 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 (Mr 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 form (Mr 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 greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees 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.

Mutation of Residue 33 of Human Equilibrative Nucleoside Transporters 1 and 2 Alters Sensitivity to Inhibition of Transport by Dilazep and Dipyridamole

“…The transport of [ 3 H]uridine (Moravek Biochemicals, Brea, CA) by logarithmically proliferating yeast was measured as described previously using the "oil stop" method (30,31) with the following modifications. Yeast were grown in CMM/GLU to an A 600 of 0.7-1.5, washed once with fresh medium, and resuspended to an A 600 of 2.0 in fresh medium.…”

Nucleoside Transporter Proteins of Saccharomyces cerevisiae

“…Nucleoside Uptake in Yeast-The uptake of 3 H-nucleosides (purchased from Moravek Biochemicals, Brea, CA) by logarithmically proliferating yeast was measured as described previously (32) using the "oil stop" method (38,39) with the following modifications. Yeast were grown in CMM/GLU or CMM/GAL to an A 600 of 0.8 -1.5, washed three times with fresh medium, and resuspended to an A 600 of 4.…”

Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes

“…1). Studies with cells grown in tunicamycin, a drug that prevents glycosylation, demonstrated that es activity is not dependent on glycosylation of the transporter protein (Hogue et al 1990). The es transport activity is thought to be ubiquitous (Cass 1995) and this conclusion is supported by the presence of hENT1 mRNA in all tissues probed thus far Pennycooke et al 2001).…”