Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: order to disorder (Structural transition)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Cholera toxin is assembled from two subunits in the periplasm of Vibrio cholerae and disassembled in the analogous compartment of target cells, the lumen of the endoplasmic reticulum (ER), before a fragment of it, the A1 chain, is transported into the cytosol. We show that protein disulfide isomerase (PDI) in the ER lumen functions to disassemble and unfold the toxin once its A chain has been cleaved. 
We designed an assay that mimics the postulated disassembly of the toxin in vivo: after arrival in the ER, the B subunits should remain bound to the ganglioside GM1 in the membrane, while the A1 fragment of the toxin should be dissociated. In our assay, membrane bound ganglioside is replaced by GM1 covalently bound to polystyrene beads. Upon incubation with ER proteins, the B subunits are expected to stay with the beads and the A1 chain to be released. The A and B subunits of the toxin were expressed in V. cholerae and the holotoxin was purified (Rodighiero et al., 1999). Due to the presence of high levels of secreted proteases in the growth medium of V. cholerae, most of the toxin molecules (about 90%) had their A subunit cleaved into the A1 and A2 fragments. The purified, nicked holotoxin was bound to GM1-coated beads and incubated under different conditions. 
To test whether the holotoxin is dissociated by ER proteins, we prepared a lumenal extract from canine microsomes and incubated it with the toxin beads. A significant fraction of the A1 subunit was found to be released into the supernatant after collection of the beads (lanes 4 and 6). 
Next, we investigated whether the release of the A1 subunit was accompanied by its unfolding. To this end, we developed a protease protection assay. Addition of high concentrations of trypsin to the folded holotoxin left all polypeptide species intact (Figure 1B, lane 2 versus 1). However, after the addition of an extract derived from the ER lumen, the A and A1 subunits were degraded whereas the B subunit remained unaffected (lanes 4 and 6). Reduction of the disulfide bridge in the A subunit was insufficient to cause trypsin sensitivity (lane 2 versus 1). 
Our results provide insight into the pathway of cholera toxin from its synthesis in bacteria up to its arrival in the lumen of the ER in mammalian cells, where its A1 fragment is released from the rest of the toxin and unfolded in preparation for its translocation into the cytosol. 
Our results explain why cholera toxin is assembled and folded in the periplasm of bacteria and disassembled and unfolded in the ER, even though both compartments normally support the folding of polypeptides. One decisive factor is that the A subunit needs to be cleaved into the A1 and A2 fragments for unfolding to occur. The periplasm of bacteria seems to lack proteases that cleave the A chain, and in this compartment, the two subunits can therefore assemble and fold into the native holotoxin.  Once in the ER, PDI binds to the A1 chain and causes it to unfold, but it leaves the ring of B chains intact.