The major exoglucanase secreted by Saccharomyces cerevisiae as a model to study protein glycosylation.
The major yeast exoglucanase (ExgIb) consists of a 408 amino acid polypeptide carrying two short N-linked oligosaccharides attached to asparagines 165 (Asn(165)) and 325 (Asn(325)). These oligosaccharides are very similar, in both length and composition, to those present in the vacuolar protease carboxypeptidase Y. Minor glycoforms of exoglucanase arise by underglycosylation of the protein precursor (Exg(165) and Exg(325)) or by elongation of the second oligosaccharide (ExgIa). The fact that these glycoforms can be readily separated and identified by HPLC and/or Western blots converts ExgI in an excellent model to study the role of the several components or branches of the precursor oligosaccharide in the efficiency and selectivity of the oligosaccharidyl transferase in vivo. We have found that the presence of a single glucose attached to Dol-PP-GlcNAc(2)-Man(9) increases the efficiency of transfer of that oligosaccharide to the protein acceptor. Also, the glucotriose unit appears to be involved in the selection of the sequons to be occupied, in such a way that its absence results in a bias towards the glycosylation of a particular sequon. Finally, we have shown the transfer of GlcNAc(2) from Dol-PP-GlcNAc(2) to exoglucanase, an indication that this intermediate is able to translocate from the cytoplasmic to the lumenal face of the endoplasmic reticulum membrane.
Larriba
G
G
Departamento de Microbiología, F. de Ciencias, Universidad de Extremadura, 06071, Badajoz, Spain. glarriba@unex.es
Cueva
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D001216:Asparagine / Q000032:analysis; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000737:chemistry*; D015698:Genomic Library; D043326:Glucan 1,3-beta-Glucosidase; D005947:Glucose / Q000032:analysis; D006031:Glycosylation; D006602:Hexosyltransferases; D007700:Kinetics; D008565:Membrane Proteins; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014166:Transferases / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism
D005656:Fungal Proteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D001216:Asparagine; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; D005947:Glucose