The oligosaccharyltransferase has been purified from Saccharomyces cerevisiae as an hetero-oligomeric complex composed of four or six subunits. Here, the in vivo subunit composition and stoichiometry of the oligosaccharyltransferase were investigated by attaching an epitope coding sequence to a previously characterized subunit gene, OST3. Five (Ost1p, Wbp1p, Swp1p, Ost2p, and Ost5p) of the seven polypeptides that were coimmunoprecipitated with the epitope-tagged Ost3p were identical to those obtained by the conventional purification procedure. Two additional coprecipitating polypeptides with apparent molecular masses of 60 and 3.6 kDa were identified as the 78-kDa Stt3 protein and the 36-residue Ost4 protein, respectively. Stt3p and Ost4p were previously identified in screens for gene products involved in N-linked glycosylation. Quantification of the in vivo radiolabeled subunits and the radioiodinated purified enzyme shows that the yeast oligosaccharyltransferase is composed of equimolar amounts of eight subunits. Exposure of the immunoprecipitated oligosaccharyltransferase to mild protein denaturants yielded a subcomplex comprised of Stt3p, Ost3p, and Ost4p. These experiments, taken together with genetic and biochemical evidence for subunit interactions, suggest that the enzyme is composed of the following three subcomplexes: (a) Stt3p-Ost4p-Ost3p, (b) Swp1p-Wbp1p-Ost2p, and (c) Ost1p-Ost5p.N-Glycosylation of proteins is an essential, highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST) 1 catalyzes the transfer of a preassembled high mannose oligosaccharide (Glc 3 Man 9 GlcNAc 2 ) onto Asn-X-Ser/Thr acceptor sites on nascent polypeptides as they are translocated into the lumen of the rough endoplasmic reticulum (for reviews see Refs. 1, and 2). Biochemical, molecular biological, and genetic studies have led to the identification of a surprisingly large number of proteins that are required for the expression of wild-type OST activity.The yeast OST was initially purified as an oligomeric complex composed of six subunits that are designated as Ost1p (62/64 kDa), Wbp1p (45 kDa), Ost3p (34 kDa), Swp1p (30 kDa), Ost2p (16 kDa), and Ost5p (9 kDa) (3). However, catalytically active tetrameric OST complexes that appear to lack Ost2p and Ost5p have also been described (4, 5). In addition, the 34-kDa Ost3 protein appears to be present in reduced amounts relative to the other three subunits in the purified OST tetramer (4), raising the possibility that oligomeric forms of the OST may exist that differ with respect to the presence of regulatory or accessory subunits.Prior to purification of the yeast enzyme, genetic and biochemical studies had established that Wbp1p and Swp1p were essential for in vivo and in vitro expression of OST activity (6, 7). Mutations in genes encoding Ost1p, Ost2p, Ost3p, and Ost5p also cause substantial reductions in both the N-linked glycosylation of proteins in vivo and the transfer of dolichollinked oligosaccharides to ac...