THE ALPHA-SUBUNIT OF THE SACCHAROMYCES-CEREVISIAE OLIGOSACCHARYLTRANSFERASE COMPLEX IS ESSENTIAL FOR VEGETATIVE GROWTH OF YEAST AND IS HOMOLOGOUS TO MAMMALIAN RIBOPHORIN-I

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins i n the lumen of the rough endoplasmic reticulum. The Saccharomyces cere visiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltr ansferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha s ubunit of the oligosaccharyltransferase. Protein sequence analysis rev ealed a significant sequence identity between the Saccharomyces cerevi siae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 loc us was not tolerated in haploid yeast showing that expression of the O st1 protein is essential for vegetative growth of yeast. An analysis o f a series of conditional ost1 mutants demonstrated that defects in th e Ost1 protein cause pleiotropic underglycosylation of soluble and mem brane-bound glycoproteins at both the permissive and restrictive growt h temperatures. Microsomal membranes isolated from ost1 mutant yeast s how marked reductions in the in vitro transfer of high mannose oligosa ccharide from exogenous lipid-linked oligosaccharide to a glycosylatio n site acceptor tripeptide. Microsomal membranes isolated from the ost 1 mutants contained elevated amounts of the Kar2 stress-response prote in.