Studies on the function of oligosaccharyl transferase subunits: A glycosylatable photoprobe binds to the luminal domain of Ost1p

Abstract

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...

Cell Wall Integrity Signaling inSaccharomyces cerevisiae

“…Specifically, early in the cell cycle, Pkc1 was detected at the prebud site and at bud tips, a pattern that is very similar to that of Rho1 (265,359). Later in the cell cycle, it becomes delocalized and finally relocalized at the mother-bud neck.…”

“…Moreover, it seems likely that Rho1 coordinates these functions at the cell surface. It is localized to sites of polarized growth in a manner dependent on the actin cytoskeleton (16,265,359).…”

Unraveling the Mechanism of Protein N-Glycosylation

“…Both Ost1p and Wbp1p have short cytosolic tails, and deletion of this tail has no effect on the growth phenotype of the yeast cell, indicating they are not essential for the function of Ost1p and Wbp1p (19,20). With respect to their transmembrane domains, the specific transmembrane sequence of Ost1p is not required for its function; instead, it appears only to be important in anchoring the protein to the ER membrane (19). In contrast, the specific sequences of the transmembrane domains in Ost4p and Wbp1p play a critical role in mediating their physical association with their partners.…”

“…As a result, Ost1p was suggested to be the subunit that is responsible for peptide substrate binding. However, after the covalent attachment site in Ost1p was narrowed down to a 9 amino acid segment (19), it was found that various mutations on this region did not cause significant defects in photolabeling or glycosylation of membrane bound and soluble glycoproteins in vivo suggesting that the luminal part of Ost1p is in close proximity to the substrate binding and/or the catalytic site, but is not directly involved in substrate recognition (19). Rather, Stt3p emerged as a strong candidate to be directly involved in the recognition/catalysis process based on subsequent photo-labeling studies in which synthetic peptides which contain several photo-reactive groups in the form of benzoylphenyllanine residues at various positions were utilized as the acceptor peptides (34).…”

Ribophorin I acts as a substrate-specific facilitator of N-glycosylation

“…Ribophorin I was identified as a subunit of the mammalian OST well before the discovery of STT3 and was initially proposed to recruit the dolichol-bound glycan to the OST . Later studies suggested that ribophorin I might form part or all of the OST active site (Yan et al, 1999), although this now seems unlikely (Yan and Lennarz, 2002a). Nevertheless, some role for ribophorin I in the process of N-glycosylation is supported by its presence in all three mammalian OST isoforms that display catalytic activity , and studies of its S. cerevisiae equivalent, Ost1p, where conditional mutants show reduced levels of glycosylation (Silberstein et al, 1995).…”