# Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC
## 3 Discussion
Whether reduction of the disulfide bond in ricin is coupled to its translocation to the cytosol is not known, and it is possible that the protection afforded by EACC could directly affect the translocation machinery in the ER as well as the reduction of the disulfide bond. It was previously reported that inhibition of transport out of the ER by expression of a negative Sar1 mutant protected against ricin [16], but whether the release of the A-chain was also affected was not investigated at that time. It was also suggested [17] that interfering with ER kinases that regulate transport out of the ER might affect the translocation of protein toxins to the cytosol. The tyrosine kinase LTK was reported to interact with Sec12, a GEF for Sar1, and thereby serve as a regulator of ER exitsite biogenesis [2; 14]. However, the inhibitor of LTK, crizotinib, which was shown to retard transport out of the ER [14], did not inhibit the toxicity of ricin nor did it have any effect on the protection afforded by EACC. Similarly, addition of 8-Br-cAMP, which also affects ER signaling and transport [13], had no effect on intoxication with ricin. A compound reported to inhibit transport of syntaxin 5 out of the ER is Retro-2 [18; 19]. However, the action of EACC seems to be different from that of Retro-2 as EACC does not protect against ricin in the presence of serum, and under serum starvation, the protection against ricin is very good, in contrast to what is seen with Shiga toxin, results which are opposite for Retro-2. Retro-2 was found to protect strongly against Shiga toxin by preventing movement of the Shiga toxin binding protein GPP130 from endosomes to the Golgi apparatus [18]. In the case of EACC, there did not seem to be any change in colocalization of syntaxin 5 with GM130, and there also was no change in colocalization of GPP130 with TGN46 (our unpublished results). The protection afforded by EACC is not expected to be related to Stx17, as it was reported that depletion of Stx17 did not affect neither anterograde transport of VSVG-GFP nor retrograde transport of VSVG-KDEL-R-YFP [10]. However, there may be an effect on exit of other proteins that might be important for reduction and translocation of ricin to the cytosol. Stx17 interacts with p24 proteins, proteins known to interact with cargos leaving the ER [10]. Additionally, Stx17 is required for a normal structure of the ERGIC and the Golgi apparatus. It cannot be excluded that structural changes of the ER in themselves might change the interaction of ricin with endogenous proteins involved in its reduction and cytosolic entry [20]. Both ricin and Shiga toxin have been reported to interact with Sec61 [21; 22; 23; 24]. However, since there was such a large protection against ricin but essentially no protection against Shiga toxin, one would not expect that Sec61 was the target of EACC. In agreement with that, immunolabeling of Sec61\(\alpha\) (and also BIP) did not reveal any large changes (our unpublished results). Now that the cellular localization for interference with ricin entry caused by EACC seems to be localized to the ER, future studies can be concentrated on the ER proteins possibly affected. An increased understanding of EACC-induced changes can provide us with a better understanding of regulation of transport in the ER.