title: C-terminal extension of phaseolin with a short methionine-rich sequence can inhibit trimerisation and result in high instability.
abstract: In an attempt to increase the content in essential amino acids methionine and tryptophan of the trimeric storage protein phaseolin, we fused a Met- and Trp-rich sequence to the C-terminus of a phaseolin variant lacking its vacuolar sorting signal, with the aim to target the protein for secretion and accumulation into the apoplast. The fate of the mutant protein, denominated Y3, was studied in transiently transfected tobacco protoplasts. We report that the presence of the additional sequence causes structural defects which inhibit trimerization and lead to partial aggregation of Y3. The protein interacts with the ER chaperone BiP prior to being degraded very rapidly, in a process that does not require vesicular transport from the ER. The rate of degradation of Y3 is higher than that observed for another assembly defective mutant of phaseolin, delta360, which remains monomeric and does not aggregate. This indicates that the plant ER quality control machinery can dispose of defective proteins with different kinetics and perhaps mechanisms, depending on the nature of their defect.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: James
authors.initials: J
authors.lastname: Nuttall
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitale
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Frigerio
mesh_terms: D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D008715:Methionine / Q000235:genetics*; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology; D014162:Transfection
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C048826:phaseolin protein, Phaseolus vulgaris; D020126:Brefeldin A; D008715:Methionine