title: Ribosome-mediated folding of partially unfolded ricin A-chain.
abstract: After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.
authors.affiliation: School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: R H
authors.initials: RH
authors.lastname: Argent
authors.forename: A M
authors.initials: AM
authors.lastname: Parrott
authors.forename: P J
authors.initials: PJ
authors.lastname: Day
authors.forename: L M
authors.initials: LM
authors.lastname: Roberts
authors.forename: P G
authors.initials: PG
authors.lastname: Stockley
authors.forename: J M
authors.initials: JM
authors.lastname: Lord
authors.forename: S E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D001483:Base Sequence; D011487:Protein Conformation; D017510:Protein Folding; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
chemical_list: D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D012276:Ricin