Ribosome-mediated folding of partially unfolded ricin A-chain.
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.
Argent
R H
RH
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Parrott
A M
AM
Day
P J
PJ
Roberts
L M
LM
Stockley
P G
PG
Lord
J M
JM
Radford
S E
SE
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
D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D012276:Ricin