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A screen for yeast mutants with defects in the dolichol-mediated pathway for N-glycosylation

Roos, J., Sternglanz, R., Lennarz, W.J.
Proceedings of the National Academy of Sciences of the United States of America 1994 v.91 no.4 pp. 1485-1489
Saccharomyces cerevisiae, mutants, mutagenesis, glycosyltransferases, structural genes, chemical reactions, glycoproteins, oligosaccharides
Dolichol in the form of dolichyl phosphate participates in the synthesis of N- and O-linked glycoproteins and phosphatidylinositol-linked proteins in the yeast Saccharomyces cerevisiae. In this organism, as well as in higher eukaryotes, a number of the enzymes in the polyisoprenoid and glycoprotein biosynthetic pathways have not been identified. In this study, we have developed a convenient, highly sensitive assay that uses one of the end products of the dolichyl-phosphate synthetic pathway, oligosaccharide-diphosphodolichol, and a (125)I-labeled peptide substrate for N-linked glycosylation to screen a collection of temperature-sensitive yeast mutants for defects in protein glycosylation. By using a combination of biochemical and genetic procedures, the defective mutants were grouped into three categories: those containing defects in dolichyl-phosphate synthesis (class 1), lipid-linked oligosaccharide assembly (class 2), or oligosaccharide transferase activity (class 3). Among the mutants identified by this screen were sec59 (which encodes dolichol kinase) and a mutant that affects the activity of the ALG1-encoded mannosyltransferase that forms dolichol-PP-(GlcNAc)(2)Man(1). Of particular interest was a mutant that exhibits a temperature-sensitive defect in oligosaccharide transferase activity. This mutant, meg1 (microsomal protein essential for glycosylation 1) assembles a complete oligosaccharide chain and, therefore, is likely to be a class 3 mutant. We report the cloning of MEG1, the gene that rescues the oligosaccharide transferase activity defect in this mutant. A number of criteria distinguish this gene from previously described genes in this pathway.