Abstract. Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (c~-~). The a, [3, y, and 8 PARAGINE-linked glycosylation of proteins is a highly conserved protein modification reaction that occurs in the lumen of the rough endoplasmic reticulum in all eukaryotic organisms (Kornfeld and Kornfeld, 1985;Herscovics and Orlean, 1993). The initial stage in the biosynthesis of N-glycosylated proteins, catalyzed by the lumenally oriented enzyme oligosaccharyltransferase, involves the transfer of a preassembled high-mannose oligosaccharide (Glc3Man9GlcNAc2) from a dolichol-pyrophosphate donor onto asparagine acceptor sites within the consensus sequon Asn-X-Ser/Thr, where X can be any amino acid except proline (Gavel and Von Heijne, 1990). N-linked glycosylation is an obligatory event for the efficient folding and oligomeric assembly of many nascent

Abstract. 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 in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (a-D, two of which are glycoproteins (a and B)-The B and 8 subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OS/7 gene that encodes the ot subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ostl protein and ribophorin I, a previ.ously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OS'H locus was not tolerated in haploid yeast showing that expression of the Ostl protein is essential for vegetative growth of yeast. An analysis of a series of conditional ostl mutants demonstrated that defects in the Ostl protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ostl mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ostl mutants contained elevated amounts of the Kar2 stress-response protein.PARAGINE-Iinked glycosylation of proteins is a ubiquitous protein modification reaction in eukaryotic organisms that occurs in the lumen of the rough endoplasmic reticulum (Herscovics and Orlean, 1993;Kornfeld and Kornfeld, 1985). Addition of asparagine-linked carbohydrates to many glycoproteins is an obligatory event for folding and assembly of newly synthesized polypeptides (Helenius, 1994). The presence of oligosaccharides is often required for the efficient transport of individual glycoproteins through the secretory pathway (Guan et al., 1985;Riederer and Hinnen, 1991;Winther et al., 1991). Glycan groups contribute to the overall dynamic stability of proteins, in some cases rendering them more resistant to proteolysis in vivo (Barriocanal et al., 1986). Diverse biological roles for asparagine-linked oligosaccharides have been identified including serving as receptors for extracellular

Abstract. We have used proteinase K as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behavior of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane.

. We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome . Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent . The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A. J., and D. 1. Meyer. 1990. Nature (Lond.) . 346: 540-544) . RER membrane proteins were reconstituted

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