STT3, A HIGHLY CONSERVED PROTEIN REQUIRED FOR YEAST OLIGOSACCHARYL TRANSFERASE-ACTIVITY IN-VIVO

N-linked glycosylation is a ubiquitous protein modification, and is es sential for viability in eukaryotic cells. A lipid-linked core-oligosa ccharide is assembled at the membrane of the endoplasmic reticulum and transferred to selected asparagine residues of nascent polypeptide ch ains by the oligosaccharyl transferase (OTase) complex. Based on the s ynthetic lethal phenotype of double mutations affecting the assembly o f the lipid-linked core-oligosaccharide and the OTase activity, we hav e performed a novel screen for mutants in Saccharomyces cerevisiae wit h altered N-linked glycosylation. Besides novel mutants deficient in t he assembly of the lipid-linked oligosaccharide (alg mutants), we iden tified the STT3 locus as being required for OTase activity ill vivo. T he essential STT3 protein is similar to 60% identical in amino acid se quence to its human homologue. A mutation in the STT3 locus affects su bstrate specificity of the OTase complex in vivo and in vitro. In stt3 -3 cells very little glycosyl transfer occurs from incomplete lipid-li nked oligosaccharide, whereas the transfer of full-length Glc(3)Man(9) GlcNAc(2) is hardly affected as compared with wild-type cells. Depleti on of the STT3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of OTase complex.