N-Linked glycoprotein assembly. Evidence that oligosaccharide attachment occurs within the lumen of the endoplasmic reticulum.

The transbilayer orientation of the oligosaccharide chain transferred from oligosaccharide-lipid to endogenous protein acceptors in sealed hen oviduct microsomes has been examined using endo-beta-N-acetylglucosaminidase H as a topological probe. The oligosaccharide moiety of these acceptors was released by the enzyme only under conditions where the microsomes were made permeable to macromolecules. The release of the oligosaccharide chain by endo-beta-N-acetylglucosaminidase H was not increased by removal of ribosomes or by mild trypsinization of the sealed microsomes. The endogenous acceptors were shown to be membrane-associated proteins that are not a part of the mRNA . ribosome . tRNA . nascent chain complex. From these results we conclude that the transfer of oligosaccharide from oligosaccharide-lipid occurs at the luminal face of the rough endoplasmic reticulum.