The ALG10 locus of Saccharomyces cerevisiae encodes the alpha-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation.
The biosynthesis of the lipid-linked oligosaccharide substrate for N-linked protein glycosylation follows a highly conserved pathway at the membrane of the endoplasmic reticulum. Based on the synthetic growth defect in combination with a reduced oligosaccharyltransferase activity (wbp1), we have identified alg10 mutant strains which accumulate lipid-linked Glc2Man9GlcNAc2. We cloned the corresponding wild-type gene and show in a novel in vitro assay that Alg10p is a dolichyl-phosphoglucose-dependent glucosyltransferase which adds the terminal alpha-1,2 glucose to the lipid-linked Glc2Man9GlcNAc2 oligosaccharide. Hypoglycosylation of secreted proteins in alg10 deletion strains demonstrates that the terminal alpha-1,2-linked glucose residue is a key element in substrate recognition by the oligosaccharyltransferase. This ensures that primarily completely assembled oligosaccharide is transferred to protein.
Burda
P
P
Mikrobiologisches Institut, ETH Zürich, Switzerland.
Aebi
M
M
D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D006602:Hexosyltransferases; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases / Q000378:metabolism
D008565:Membrane Proteins; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; C113849:DIE2 protein, S cerevisiae; D005964:Glucosyltransferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase