## 2 Cellular Pathways Exploited by Toxins
### Retrograde Toxin Transport from the Golgi to the ER and Translocation to the Cytosol  
A well-studied mechanism for retrograde transport from the Golgi to the ER is the COPI-mediated transport of molecules containing a KDEL-sequence (for review, see [109; 110]). However, neither Shiga toxin nor ricin have such a sequence, but are still able to go from the trans-Golgi network to the ER, as illustrated for Shiga toxin in Figure 4. Studies with retrograde transport of Shiga toxin have elucidated the complexity of retrograde transport in general, since Shiga toxin was found to be transported to the ER by a COPI-independent, Rab6-dependent pathway [111; 112; 113]. Later studies of Shiga toxin and ricin have used siRNA or shRNA to add other candidates to the list of molecules that may be involved in retrograde transport [114; 115]. This includes COPII, TRAPP, and GARP complexes. However, it is important to be aware of that some of the effects could be indirect. For example, an incorrect sorting of the enzyme furin, involved in cleavage and activation of Shiga toxin [116], might lead to protection but may not necessarily affect toxin transport (for a discussion of these studies, see [16]). Furthermore, changes of the Golgi apparatus, for instance, disruption of this organelle due to expression of a temperature-sensitive \(\in\)-COP subunit, were found to induce an alternative BFA-resistant transport pathway for ricin to the ER, illustrating the ability of the cell to compensate for blocks in transport [117]. Several different molecules have now been identified to be involved in retrograde transport and for translocation of the enzymatically active part of the toxins (for review, see [95; 96]). Again, it is important to be aware of the ability of cells to induce compensatory processes. For instance, Sec61 was not found in a screening for molecules able to protect against ricin [115] but was previously found to be able to interact with ricin [118; 119; 120]. Consequently, one may wonder if an alternative translocation mechanism is somehow operating under those conditions. However, the screening indicated the importance of derlins, and it is possible that several molecules can support translocation to the cytosol. This is in contrast to Shiga toxin, which has also been reported to be in a complex with Sec61 [121]. Screening studies have also supported the idea that Sec61 is involved in transport to the cytosol [115]. The compound Retro-2 is an example of how identification of compounds that protect against ricin and Shiga toxin may provide us with basic knowledge about general mechanisms. This drug provides very good protection against Shiga toxin, apparently due to lack of transport of GPP130 from endosomes to the Golgi apparatus [122]. This is associated with its ability to target the ER component Sec16A and prevent transport of syntaxin 5 from ER to the Golgi [122]. However, the drug also protects against ricin [123], but the explanation for this protection is not obvious. Future studies are likely to provide more information about the relation of syntaxin 5 to other SNARE complexes and transport in the ER-Golgi area. Interestingly, Retro-2 was reported to inhibit ASNA-1 mediated ER targeting and membrane insertion of tail-anchored proteins [124], a finding that may be related to protection against ricin. Ricin transport from endosomes to the Golgi apparatus was reported to be inhibited only by 20% upon knockdown of ASNA-1 [125]. Thus, ricin  
Figure 4: Intracellular localization of endocytosed Shiga toxin-HRP in A431 cells. Shiga toxin is observed in the Golgi cisternae (GO), the endoplasmic reticulum (ER) and the nuclear envelope (NE). Scale bars are 0.5 \(\upmu\)m. Reproduced with permission from ref. [12] Copyright 1992 Springer Nature. transport seems to be blocked at a later step in the retrograde pathway than Shiga toxin. In 1995, it was reported that transient expression of mutants of GTPases Sar1, ARF1, and Rab1, protected against ricin, thereby interfering with ER-Golgi trafficking [126].