A new yeast mutation in the glucosylation steps of the asparagine-linked glycosylation pathway. Formation of a novel asparagine-linked oligosaccharide containing two glucose residues.
We have isolated and characterized a new yeast mutation in the glucosylation steps of lipid-linked oligosaccharide biosynthesis, alg8-1. Cells carrying the alg8-1 mutation accumulate Glc1Man9GlcNAc2-lipid both in vivo and in vitro. We present evidence showing that the alg8-1 mutation blocks addition of the second alpha 1,3-linked glucose. alg8-1 cells transfer Glc1Man9GlcNAc2 to protein instead of the wild type oligosaccharide, Glc3Man9GlcNAc2. Pulse-chase studies indicate that the Glc1Man9GlcNAc2 transferred is processed more slowly than the wild type oligosaccharide. The yeast mutation gls1-1 lacks glucosidase I activity (Esmon, B., Esmon, P.C., and Schekman, R. (1984) J. Biol. Chem. 259, 10322-10327), the enzyme responsible for removing the alpha 1,2-linked glucose residues from protein-linked oligosaccharides. We demonstrate that gls1-1 cells contain glucosidase II activity (which removes alpha 1,3-linked glucose residues) and have constructed the alg8-1 gls1-1 haploid double mutant. The Glc1Man9GlcNAc2 oligosaccharide was trimmed normally in these cells, demonstrating that the alg8-1 oligosaccharide contained an alpha 1,3-linked glucose residue. A novel Glc2 compound was probably produced by the action of the biosynthetic enzyme that normally adds the alpha 1,2-linked glucose to lipid-linked Glc2Man9GlcNAc2. This enzyme may be able to slowly add alpha 1,2-linked glucose residue to protein-bound Glc1Man9GlcNAc2. The relevance of these findings to similar observations in other systems where glucose residues are added to asparagine-linked oligosaccharides and the possible significance of the reduced rate of oligosaccharide trimming in the alg mutants are discussed.
D001216:Asparagine / Q000378:metabolism*; D002236:Carbohydrate Conformation; D004287:Dolichol Monophosphate Mannose / Q000096:biosynthesis; D005947:Glucose / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D007700:Kinetics; D050356:Lipid Metabolism; D008358:Mannose / Q000378:metabolism; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000235:genetics; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D006023:Glycoproteins; D009844:Oligosaccharides; D011103:Polyisoprenyl Phosphate Monosaccharides; D004287:Dolichol Monophosphate Mannose; C002968:dolichol-D-glucosylmonophosphate; D001216:Asparagine; D005947:Glucose; D008358:Mannose