title: Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.
abstract: The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.
authors.affiliation: Biological Laboratory, University of Kent, Canterbury, UK.
authors.forename: N J
authors.initials: NJ
authors.lastname: Bulleid
authors.forename: R B
authors.initials: RB
authors.lastname: Freedman
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002474:Cell-Free System; D004285:Dogs; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism; D007535:Isomerases / Q000502:physiology*; D000072235:Mating Factor; D008861:Microsomes / Q000378:metabolism*; D010179:Pancreas; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D019704:Protein Disulfide-Isomerases; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins
chemical_list: D006023:Glycoproteins; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D000072235:Mating Factor; D007371:Interferon-gamma; D007535:Isomerases; D019704:Protein Disulfide-Isomerases