title: Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon.
abstract: The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: Cornelia M
authors.initials: CM
authors.lastname: Wilson
authors.forename: Claudine
authors.initials: C
authors.lastname: Kraft
authors.forename: Claire
authors.initials: C
authors.lastname: Duggan
authors.forename: Nurzian
authors.initials: N
authors.lastname: Ismail
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Crawshaw
authors.forename: Stephen
authors.initials: S
authors.lastname: High
mesh_terms: D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D003062:Codon; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012270:Ribosomes / Q000737:chemistry; D000069816:SEC Translocation Channels; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D016564:Amyloid beta-Protein Precursor; D003062:Codon; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D008565:Membrane Proteins; D010455:Peptides; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D000069816:SEC Translocation Channels; C034241:ribophorin; D013395:Sucrose; D003513:Cycloheximide; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D003545:Cysteine