GLYCOSYLATION OF YEAST EXOGLUCANASE SEQUONS IN ALG MUTANTS DEFICIENT IN THE GLUCOSYLATION STEPS OF THE LIPID-LINKED OLIGOSACCHARIDE - PRESENCE OF GLUCOTRIOSE UNIT IN DOL-PP-GLCNAC(2)MAN(9)GLC(3) INFLUENCES BOTH GLYCOSYLATION EFFICIENCY AND SELECTION OF N-LINKED SITES

The major exoglucanase (Exg) from Saccharomyces cerevisiae has a short N-linked oligosaccharide attached to each of the potential glycosylat ion sites present in the primary translation product. We have studied the Exg glycoforms secreted by alg mutants deficient in the final step s of the assembly of dolichol-P-P-GlcNAc(2)-Man(9)-Glc(3). These mutan ts synthesize and transfer to nascent proteins truncated oligosacchari des lacking two (alg8) or three (alg5 and alg6) glucoses. In addition to the enzyme carrying both sugar chains (ExgII), all three mutants se creted underglycosylated forms containing one oligosaccharide attached to either the first (ExgII'(1/2)) or the second (ExgII(1/2)) potentia l glycosylation site, and nonglycosylated enzyme (Exg(Tuni)). As compa red with alg5 and alg6, alg8 secreted a higher proportion of ExgII, wh ich was paralleled by a significant drop in the proportion of Exg(Tuni ) and, to a lesser extent, of ExgII(1/2). The presence of a single glu cose attached to Dol-P-P-GlcNAc(2)-Man(9) therefore increases the effi ciency of transfer of the that oligosaccharide to the protein acceptor in vivo. Moreover, whereas ExgII'(1/2) was never secreted by wild typ e cells, it was the most abundant underglycosylated form secreted by a h three mutants. These mutants are affected in the efficiency at which the individual sequons that are glycosylated, and this suggests a rol e for the glucotriose unit in the selection of the sequons are to be o ccupied in glycoproteins synthesized by wild type.