# Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC
## 1 Introduction  
It is becoming increasingly clear that a number of cellular processes such as endocytosis, degradation, and anterograde as well as retrograde transport in the Golgi apparatus and the ER are subject to complex regulation by signaling [1; 2]. Thus, when attempting to study regulation of one specific process by knockdown of certain proteins or by manipulation of SNARE complexes or their phosphorylation status, it is challenging to obtain a complete overview of the cellular responses and the changes induced.  
Protein toxins such as the plant toxin ricin and the bacterial toxin Shiga toxin have proven valuable to investigate changes in several intracellular transport steps [3]. These toxins consist of two moieties, one that binds to the cell surface and another moiety that inhibits protein synthesis by enzymatically removing one adenine from the 28S RNA of the 60S ribosomal subunit after entry into the cytosol [3; 4; 5; 6]. In the case of ricin, the toxin binds to both glycoproteins and glycolipids with terminal galactose, whereas Shiga toxin binds to the neutral glycosphingolipid Gb3. Both toxins are endocytosed by different endocytic mechanisms, and they are then transported to the Golgi apparatus and the ER before the enzymatically active subunit is released and translocated across the ER membrane.  
Importantly, the toxins seem to differ when it comes to regulation of their entry into cells and the retrograde transport [3]. Not only do they differ when it comes to the kinetics of retrograde transport, but also the proteins and lipid species required for transport are different, with differences that can be exploited to study the cellular processes involved and their regulation.  
It was recently reported that the compound EACC is a specific inhibitor of fusion between autophagosomes and lysosomes [7] and that this compound prevented recruitment of the SNAREs syntaxin 17 (Stx17) and SNAP29 onto the autophagosomal membrane. It also inhibited the interaction of Stx17 with the HOPS subunit Vps33A and the SNARE VAMP8. Furthermore, the TANK-binding kinase 1 (TBK1) controls autophagy initiation by binding to and phosphorylating Stx17 [8]. Since Stx17 has also been reported to be present in the ER [9], the ERGIC [10], and the Golgi apparatus [8; 10], and to be involved in ER dynamics [9], we wanted to investigate whether the compound EACC and an inhibitor of TBK1, MRT68601 [11], affects retrograde toxin transport and intoxication. EACC might be an important tool also to investigate ER function, although its exact target is not yet known.  
As demonstrated in the current article, EACC can provide a very good protection against the plant toxin ricin, a protection which seems to be related to the inhibited release of ricin A-chain after toxin entry into the ER. Unexpectedly, the TBK1 inhibitor strongly increased ricin transport to the Golgi apparatus, sensitized the cells to ricin, and thereby counteracted the protective effect of EACC. However, EACC was still able to protect against ricin in the presence of MRT68601. EACC protected different cell types also against other plant toxins, such as abrin, modeccin, viscumin, and volkensin, demonstrating that it may have a general effect on translocation of plant toxins from the ER. In contrast, there was only a very small protection on the effect of Shiga toxin, supporting the view that the cellular mechanisms for entry are not identical.