Abstract
N-linked oligosaccharides have been examined on glycoproteins accumulated in yeast mutants that are blocked at successive stages in the secretory pathway, and in a new mutant, gls1-1, deficient in removal of glucose from N-linked core oligosaccharides, but not blocked in secretion. Oligosaccharides on invertase, a secreted protein, and carboxypeptidase Y, a vacuolar protein, are matured normally in the gls1 mutant but retain three glucoses/carbohydrate chain. The gls1 mutation is recessive and extracts of mutant cells are inactive in release of labeled glucose from core oligosaccharides. The mutant thus lacks glucosidase I activity but could also be deficient in the other core oligosaccharide glucosidase. When transport from the endoplasmic reticulum is blocked in sec18, N-linked oligosaccharides accumulate with a size corresponding to Man8GlcNAc2 when the normal GLS1 allele is present, and Glc3Man8GlcNAc2 in the gls1 mutant. From this we infer that all glucose units are removed prior to glycoprotein transport from the endoplasmic reticulum.
Highlights
Glycoproteins accumulated in yeast mutants that are In thecourse of our studies onthe stage-specificprocessing blocked at successive stages in the secretory pathway, of oligosaccharides in yeast secretory mutants, we found and in a new mutant, glsl-1, deficient in removal of a mutant that produced glycoproteins with slightly glucose from N-linked core oligosaccharides, but not larger oligosaccharide chains
The gkl-1 and GlcsManaGlcNAc2 in the glsl mutant. From this mutation was detected by a reduction in SDS gel mobility of carboxwe infer that all glucose units are removed prior to glycoprotein transport from the endoplasmic reticulum
Mutant strainslack glucosidase I activity, and oligosaccharides retain all threeglucoses on glycoproteins transported to thveacuole and to thecell surface
Summary
Characteristics of the N-linked oligosaccharides, present on glycoproteins accumulated in the ER of secretion-blocked mutant strains, arepresented. The gkl-1 and GlcsManaGlcNAc2 in the glsl mutant From this mutation was detected by a reduction in SDS gel mobility of carboxwe infer that all glucose units are removed prior to glycoprotein transport from the endoplasmic reticulum. For this reason, the influence of Labeling, Extraction, and Characterization of Mannoproteins-Dur- the glsl-1mutation was assessed with invertase accumulated ing [3H]mannoselabeling, cells were grownin YPD medium containing 0.1% glucose, and labeled with 200 pCi of [3H]mannose. The glsl-1mutation resulted in tion with ethanol; oligosaccharides were released by digestion with 125 ng of endo H in 0.27 M citrate buffer (pH 5.5), 0.1% SDS in 100 pl for -12 h at 37 "C. Radiolabeled oligosaccharides were visulaized by fluorography after saturation of the plate with 0.5% 2,5-diphenyloxazole in 1-methylnapthalene
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