Oligosaccharyl transferase (OT) is a complex multisubunit enzyme that, in the case of Saccharomyces cerevisiae, contains nine different transmembrane proteins. One of our goals is to identify the OT subunit(s) responsible for recognizing the consensus sequence, -Asn-X-Thr͞Ser-, and catalyzing the oligosaccharide transfer reaction. By using a substrate-based photoprobe, earlier we found that Ost1p was specifically linked to the radiolabeled photoprobe. We have now examined Ost1p in more detail. Deletion of the cytoplasmic tail of Ost1p caused no defects in growth and glycosylation. In addition, replacement of the transmembrane domain with other hydrophobic amino acids did not impair growth. In contrast, a construct containing only the luminal domain of Ost1p did not support cell growth. Given these observations, we concentrated on studying the luminal domain of Ost1p and localized the photoprobe attachment region within a sequence of nine amino acid residues. Because mutations in the photoprobe attachment region did not cause any severe growth or glycosylation defects, we conclude that this region is not involved in the recognition of the N-glycosylation site. By further mutagenesis of the conserved residues of Ost1p we conclude that the luminal domain mediates interactions with other subunits of OT and becomes labeled because of its proximity to the recognition and͞or catalytic subunit in the OT complex, Stt3p.T he enzyme catalyzing N-linked glycosylation, called oligosaccharyl transferase (OT), is a remarkably complex multisubunit enzyme that, in the case of Saccharomyces cerevisiae, is composed of nine different transmembrane proteins (1-16). Even though genes encoding the nine subunits of yeast OT have been cloned, at present there is very limited information about the function of each of the subunits. We have undertaken the study of the mode of interaction of these transmembrane protein subunits within the endoplasmic reticulum to elucidate their function.To identify the subunit(s) of yeast OT that recognizes -Asn-X-Thr͞Ser-sites that can be glycosylated (17-19), we developed photoaffinity probes containing a photoreactive benzophenone derivative, p-benzoylphenylalanine (Bpa), as part of an 125 Ilabeled acceptor tripeptide, 125 I-Bolton-Hunter (bh)-Asn-BpaThr-Amide. By using this 125 I-labeled Bpa containing tripeptide, which was shown to be a substrate of OT, we found that after photoactivation of yeast microsomes, Ost1p was specifically labeled (20).Our current objective was to localize the site of binding of the photoprobe and to determine whether Ost1p is the actual subunit that recognizes the N-glycosylation consensus sequence -Asn-X-Thr͞Ser-. Three models to explain probe attachment to Ost1p were considered (Fig. 1). In model I, interaction of the Asn and Thr residues of the photolabeled tripeptide with the putative N-glycosylation site recognition domain occurs, and the photoreactive middle amino acid, Bpa, covalently binds to some nearby amino acid residue in the Ost1p backbone. Alternatively, in mod...