NEW PHENOTYPE OF MUTATIONS DEFICIENT IN GLUCOSYLATION OF THE LIPID-LINKED OLIGOSACCHARIDE - CLONING OF THE ALG8 LOCUS

Glc(3)Man(9)GlcNAc(2) is the preferred substrate of the oligosaccharyl transferase of N-linked glycosylation of proteins, but nonglucosylated oligosaccharides can be transferred to proteins in Saccharomyces cere visiae. Mutations affecting the addition of the three terminal glucose residues lead to accumulation of Man(9)GlcNAc(2) or Glc(1)Man(9)GlcNA c(2) in vivo but do not show any detectable growth defect. When these mutations were introduced into a strain with reduced oligosaccharyltra nsferase activity (due to the wbp1-1 mutation), a severe growth defect was observed: accumulation of suboptimal lipid-linked oligosaccharide and reduced oligosaccharyltransferase activity resulted in a severe u nderglycosylation of secreted proteins. This new synthetic phenotype m ade it possible to isolate the ALG8 locus, encoding a potential glucos yltransferase of the endoplasmic reticulum. The ALG8 protein is a 63.5 -kDa hydrophobic protein that is not essential for the vegetative grow th of yeast. However, the lack of this protein resulted in underglycos ylation of secreted proteins.