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.
D000818:Animals; D002250:Carbon Radioisotopes; D002645:Chickens; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis*; D007425:Intracellular Membranes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis; D010057:Oviducts / Q000378:metabolism*; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000138:chemical synthesis*; D011105:Polyisoprenyl Phosphate Sugars / Q000138:chemical synthesis*; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D002250:Carbon Radioisotopes; D006023:Glycoproteins; D009844:Oligosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine; C007292:P'-di-N-acetylchitobiosyl-P(2)-dolichylpyrophosphate