N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast exoglucanase.
Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-beta-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum.
Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
R
R
Cueva
C
C
Cotano
G
G
Larriba
D000581:Amidohydrolases; D001345:Autoradiography; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A / Q000378:metabolism; D005690:Galactose; D043326:Glucan 1,3-beta-Glucosidase; D006031:Glycosylation; D006596:Hexosaminidases; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D011104:Polyisoprenyl Phosphate Oligosaccharides; D003208:Concanavalin A; C016418:dolichyl-diphosphate-di-N-acetylchitobiose; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase; D006596:Hexosaminidases; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; D000581:Amidohydrolases; D005690:Galactose