Ribosome-mediated folding of partially unfolded ricin A-chain

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is t ransported to the endoplasmic reticulum, whereupon the A-chain must cross t he lumenal membrane to reach its ribosomal substrates. It is assumed that m embrane traversal is preceded by unfolding of ricin A-chain, followed by re folding in the cytosol to generate the native, biologically active toxin. H ere we describe biochemical and biophysical analyses of the unfolding of ri cin A-chain and its refolding in vitro. We show that native ricin A-chain i s surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 deg rees C to generate a partially unfolded state, This species has conformatio nal properties typical of a molten globule, and cannot be refolded to the n ative state by manip ulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosom al RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regain s full catalytic activity. The data suggest that the conformational stabili ty of ricin A-chain is ideally poised for translocation from the endoplasmi c reticulum, Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell dea th.