Transmembrane assembly of N-linked glycoproteins. Studies on the topology of saccharide synthesis.

The mechanism of synthesis of dolichol-linked saccharides has been examined using sealed hen oviduct microsomes. N,N'-Diacetylchitobiosylpyrophosphoryldolichol (chitobiosyl-lipid) was shown to be inaccessible to a soluble galactosyltransferase during its synthesis from UDP-GlcNac. In contrast, exogenous chitobiosyl-lipid added to microsomes is accessible to galactosyltransferase and does not appear to be unidirectionally translocated to the lumen. These results imply that assembly of chitobiosyl-lipid does not occur on the cytoplasmic face of the rough endoplasmic reticulum followed by a rapid unidirectional translocation to the lumen. Two lines of evidence rule out sugar nucleotide uptake and utilization within the lumen as a mechanism for chitobiosyl-lipid synthesis. GDP-mannose, which is involved in the elongation of chitobiosyl-lipid to form oligosaccharide-lipid, also does not permeate microsomal vesicles. This observation regarding GDP-mannose raises two important questions. 1) What is the topology of the oligosaccharide in sealed microsomes? and 2) how is the chitobiosyl-lipid elongated by addition of mannosyl units to form an oligosaccharide-lipid? During the synthesis of oligosaccharide-lipid in freeze-thawed microsomes, approximately 40% of the oligosaccharide is released from the lipid by hydrolysis and remains entrapped within the sealed microsomes. These data suggest that oligosaccharide-lipid is transiently present at the luminal face of the rough endoplasmic reticulum during its synthesis. Most of the enzymes involved in the synthesis of oligosaccharide-lipid can be inactivated by external proteolysis of microsomal vesicles. These data are discussed in terms of a model in which synthesis of chitobiosyl-lipid and its elongation to oligosaccharide-lipid are coupled processes that occur in a transmembrane multienzyme complex.