Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway

• Published in: The Journal of biological chemistry 1984-01, Vol.259 (1), p.412-417
• Main Authors: Runge, K W, Huffaker, T C, Robbins, P W
• Format: Article
• Language: English
• Subjects: Acetylglucosaminidase - metabolism; Analytical, structural and metabolic biochemistry; Asparagine - metabolism; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Glycoproteins; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Mutation; Polyisoprenyl Phosphate Monosaccharides - metabolism; Polyisoprenyl Phosphate Oligosaccharides - biosynthesis; Polyisoprenyl Phosphate Sugars - biosynthesis; Proteins; Saccharomyces cerevisiae - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)43676-3

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.