Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae.
Glycosyl phosphatidylinositol (GPI) anchoring, N glycosylation, and O mannosylation of protein occur in the rough endoplasmic reticulum and involve transfer of precursor structures that contain mannose. Direct genetic evidence is presented that dolichol phosphate mannose (Dol-P-Man) synthase, which transfers mannose from GDPMan to the polyisoprenoid dolichol phosphate, is required in vivo for all three biosynthetic pathways leading to these covalent modifications of protein in yeast cells. Temperature-sensitive yeast mutants were isolated after in vitro mutagenesis of the yeast DPM1 gene. At the nonpermissive temperature of 37 degrees C, the dpm1 mutants were blocked in [2-3H]myo-inositol incorporation into protein and accumulated a lipid that could be radiolabeled with both [2-3H]myo-inositol and [2-3H]glucosamine and met existing criteria for an intermediate in GPI anchor biosynthesis. The likeliest explanation for these results is that Dol-P-Man donates the mannose residues needed for completion of the GPI anchor precursor lipid before it can be transferred to protein. Dol-P-Man synthase is also required in vivo for N glycosylation of protein, because (i) dpm1 cells were unable to make the full-length precursor Dol-PP-GlcNAc2Man9Glc3 and instead accumulated the intermediate Dol-PP-GlcNAc2Man5 in their pool of lipid-linked precursor oligosaccharides and (ii) truncated, endoglycosidase H-resistant oligosaccharides were transferred to the N-glycosylated protein invertase after a shift to 37 degrees C. Dol-P-Man synthase is also required in vivo for O mannosylation of protein, because chitinase, normally a 150-kDa O-mannosylated protein, showed a molecular size of 60 kDa, the size predicted for the unglycosylated protein, after shift of the dpm1 mutant to the nonpermissive temperature.
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Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002688:Chitinases / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D008364:Mannosyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis*; D010716:Phosphatidylinositols / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D009844:Oligosaccharides; D010716:Phosphatidylinositols; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D006026:Glycoside Hydrolases; D002688:Chitinases; D043324:beta-Fructofuranosidase