STEPWISE ASSEMBLY OF THE LIPID-LINKED OLIGOSACCHARIDE IN THE ENDOPLASMIC-RETICULUM OF SACCHAROMYCES-CEREVISIAE - IDENTIFICATION OF THE ALG9GENE ENCODING A PUTATIVE MANNOSYL TRANSFERASE

The core oligosaccharide Glc(3)Man(9)GlcNAc(2) is assembled at the mem brane of the endoplasmic reticulum on the lipid carrier dolichyl pyrop hosphate and transferred to selected asparagine residues of nascent po lypeptide chains. This transfer is catalyzed by the oligosaccharyl tra nsferase complex. Based on the synthetic phenotype of the oligosacchar yl transferase mutation wbp1 in combination with a deficiency in the a ssembly pathway of the oligosaccharide in Saccharomyces cerevisiae, we have identified the novel ALG9 gene. We conclude that this locus enco des a putative mannosyl transferase because deletion of the gene led t o accumulation of lipid-linked Man(6)GlcNAc(2) in vivo and to hypoglyc osylation of secreted proteins. Using an approach combining genetic an d biochemical techniques, we show that the assembly of the lipid-linke d core oligosaccharide in the lumen of the endoplasmic reticulum occur s in a stepwise fashion.