title: 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-ThrSer-, 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 andor catalytic subunit in the OT complex, Stt3p.
authors.affiliation: Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yan
authors.forename: William J
authors.initials: WJ
authors.lastname: Lennarz
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D016384:Consensus Sequence; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins; D009097:Multienzyme Complexes / Q000737:chemistry*; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D019920:Photoaffinity Labels / Q000032:analysis* / Q000737:chemistry; D010777:Photochemistry; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
chemical_list: C470578:125I-bolton-Hunter-asparaginyl-4-benzoylphenylalanyl-threoninamide; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D019920:Photoaffinity Labels; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase