Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Abstract

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosacchande moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [3H]glucosamine and [35S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.in the pathway of protein glycosylation is an effective strategy in elucidating the function of the glycan moiety of glycoproteins (7). One of the most specific inhibitors is TM,' a nucleoside antibiotic produced by Streptomyces lysosuperiflcus, which blocks the first step in the formation of the lipid-linked saccharide core, i.e. the transfer of GlcNAc-1-P from UDPGlcNAc to dolichyl-P to produce GlcNAc-PP-dolichyl (UDPGlcNAc:dolichyl-phosphate GlcNAc-1-P transferase) (30). In cultured soybean cells TM completely blocked the incorporation of [3H]mannose into lipid-linked oligosaccharides (14).