title: Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation.
abstract: The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins.
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Philip J
authors.identifier: 0000-0003-2576-5010
authors.initials: PJ
authors.lastname: Robinson
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Marie Anne
authors.initials: MA
authors.lastname: Pringle
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: Cheryl A
authors.identifier: 0000-0002-4774-2840
authors.initials: CA
authors.lastname: Woolhead
authors.affiliation: From the Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom neil.bulleid@glasgow.ac.uk.
authors.forename: Neil J
authors.identifier: 0000-0002-9839-5279
authors.initials: NJ
authors.lastname: Bulleid
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002474:Cell-Free System; D003062:Codon; D019295:Computational Biology; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D010179:Pancreas / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D000072417:Protein Domains; D017510:Protein Folding; D021381:Protein Transport; D001613:beta 2-Microglobulin / Q000737:chemistry
chemical_list: D003062:Codon; D003432:Cross-Linking Reagents; D004220:Disulfides; D010455:Peptides; D001613:beta 2-Microglobulin; D019704:Protein Disulfide-Isomerases
keywords: ER; disulfide; disulfide formation; endoplasmic reticulum; protein disulfide isomerase; protein folding; protein translocation; β2-microglobulin