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 Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the 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 Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains 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 branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.
Burda
P
P
Mikrobiologisches Institut, ETH Zürich, CH-8092 Zürich, Switzerland.
Jakob
C A
CA
Beinhauer
J
J
Hegemann
J H
JH
Aebi
M
M
D001483:Base Sequence; D002240:Carbohydrate Sequence; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D006031:Glycosylation; D008055:Lipids / Q000737:chemistry*; D008364:Mannosyltransferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D008055:Lipids; D009844:Oligosaccharides; D008364:Mannosyltransferases