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 oligosaccharyltransferase of N-linked glycosylation of proteins, but nonglucosylated oligosaccharides can be transferred to proteins in Saccharomyces cerevisiae. Mutations affecting the addition of the three terminal glucose residues lead to accumulation of Man(9)GlcNAc(2) or Glc(1)Man(9)GlcNAc(2) in vivo but do not show any detectable growth defect. When these mutations were introduced into a strain with reduced oligosaccharyltransferase 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 underglycosylation of secreted proteins. This new synthetic phenotype made it possible to isolate the ALG8 locus, encoding a potential glucosyltransferase of the endoplasmic reticulum. The ALG8 protein is a 63.5-kDa hydrophobic protein that is not essential for the vegetative growth of yeast. However, the lack of this protein resulted in underglycosylation of secreted proteins.