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-GlcNAc2Man9Glc3 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 glycosylation sites present in the primary translation product. We have studied the Exg glycoforms secreted by alg mutants deficient in the final steps of the assembly of dolichol-P-P-GlcNAc2-Man9-Glc3. These mutants synthesize and transfer to nascent proteins truncated oligosaccharides lacking two (alg8) or three (alg5 and alg6) glucoses. In addition to the enzyme carrying both sugar chains (ExgII), all three mutants secreted underglycosylated forms containing one oligosaccharide attached to either the first (ExgII'1/2) or the second (ExgII1/2) potential glycosylation site, and nonglycosylated enzyme (ExgTuni). As compared with alg5 and alg6, alg8 secreted a higher proportion of ExgII, which was paralleled by a significant drop in the proportion of ExgTuni and, to a lesser extent, of ExgII1/2. The presence of a single glucose attached to Dol-P-P-GlcNAc2-Man9 therefore increases the efficiency of transfer of the that oligosaccharide to the protein acceptor in vivo. Moreover, whereas ExgII'1/2 was never secreted by wild type cells, it was the most abundant underglycosylated form secreted by all three mutants. These mutants are affected in the efficiency at which the individual sequons that are glycosylated, and this suggests a role for the glucotriose unit in the selection of the sequons are to be occupied in glycoproteins synthesized by wild type.
Muñoz
M D
MD
Departmento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Hernández
L M
LM
Basco
R
R
Andaluz
E
E
Larriba
G
G
D001665:Binding Sites; D002240:Carbohydrate Sequence; D043326:Glucan 1,3-beta-Glucosidase; D005947:Glucose / Q000032:analysis; D006031:Glycosylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D001617:beta-Glucosidase / Q000378:metabolism*
C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D009844:Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; D005947:Glucose