A screen for yeast mutants with defects in the dolichol-mediated pathway for N-glycosylation.
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 dolichylphosphate synthetic pathway, oligosaccharide-diphosphodolichol, and a 125I-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)2Man1. 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.
Roos
J
J
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215.
Sternglanz
R
R
Lennarz
W J
WJ
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004288:Dolichol Phosphates / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics; D005816:Genetic Complementation Test; D006020:Glycopeptides / Q000096:biosynthesis*; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D004288:Dolichol Phosphates; D006020:Glycopeptides; D008565:Membrane Proteins; D009844:Oligosaccharides; C028306:dolichol monophosphate; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase