Cells expressing ricin B chain within the secretory pathway are significantly more resistant to intoxication by ricin holotoxin but not to other cytotoxins that exploit similar endocytic routes to the cytosol. Furthermore, cells expressing the related B chain of abrin are protected against both incoming abrin and ricin. These phenotypes can be correlated with the abilities of the respective B chains to form disulphide-linked A-B holotoxins, since abrin B chain forms heterodimers with either abrin or ricin A chains, whereas ricin B chain forms heterodimers with ricin A chain only. In the ricin B-expressing cells, this newly made lectin disappears with biphasic kinetics comprising a retention phase followed by slow turnover and disposal after disengagement from calnexin cycle components. Interference with ricin cytotoxicity occurs during the early retention phase when ricin B chain is associated with PDI (protein disulphide-isomerase). The data show that retrotranslocation of incoming toxin is impeded by PDI-catalysed formation of heterodimers between endogenous B and A chains derived from reduced holotoxin, thus proving that reduction of ricin occurs in the endoplasmic reticulum. In contrast with other toxins, ricin does not appear to require either proteolytic cleavage or unfolding for PDI-catalysed reduction.

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER‐associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL‐like C‐terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome‐inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome‐inactivating protein that can enter cells by receptor‐mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi‐independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin‐based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi‐mediated retrograde transport to reach their retrotranslocation site.

Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system but it must also cross an internal membrane and avoid complete degradation without compromising its activity in any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins having beta-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction. It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in 'magic bullets' to specifically target and destroy cancer cells, and the unusual intracellular trafficking properties of ricin potentially permit its development as a vaccine vector. Combining our understanding of the ricin structure with ways to cripple its unwanted properties (its enzymatic activity and promotion of vascular leak whilst retaining protein stability and important immunodominant epitopes), will also be crucial in the development of a long awaited protective vaccine against this toxin.