Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man 2GlcNAc 2 and Man 1GlcNAc 2 are transferred from dolichol to protein in vivo

Abstract

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man 2GlcNAc 2 synthesized by intact yeast ( Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man 2GlcNAc 2 and Man 1GlcNAc 2 (T. C. Huffaker and P. W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466–7470) . We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man 2GlcNAc 2 and Man 1GlcNAc 2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide- N 4 [ N-acetyl-β-glucosaminyl] asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 °C. Two isomers of Man 2GlcNAc 2, Man1 → 3ManGlcNAc 2 and Man1 → 6ManGlcNAc 2, were present on lipid and protein. The transfer of Man 2GlcNAc 2 and Man 1GlcNAc 2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.