Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases

The assembly of the lipid-linked core oligosaccharide Glc(3)Man(9)GlcNAc(2) , the substrate for N-linked glycosylation of proteins in the endoplasmic r eticulum (ER), is catalyzed by different glycosyltransferases located at th e membrane of the ER, We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man(7)GlcNAc(2). The biosynthesis of the highly branched oligosaccharide follows an ordered path way which ensures that only completely assembled oligosaccharide is transfe rred from the lipid anchor to proteins. Using the combination of mutant str ains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branchin g. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate spec ificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc(3)Man(9)GlcNAc(2) in the endoplasmic reticulum.