# Targeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy
## 2 Early Endosome-to-Golgi Transport of STx and STx1
### Clathrin and Retromer Coats and Accessory Proteins  
Clathrin has a well-established role in retrograde transport from early endosomes to the Golgi. STxB/STx1B was observed in clathrin-containing endosomal tubules [30], and antisense RNA against clathrin inhibits STxB/STx1B from exiting early endosomes [30; 31]. The clathrin adaptor epsinR is also found in STxB/STx1B-containing early endosomal tubules [30]. EpsinR is proposed to serve as a multivalent linker between early endosome membrane lipids and clathrin coats. Indeed, transport of STxB/STx1B from early endosomes to the Golgi is inhibited in epsinR small interfering RNA (siRNA)-treated cells [30], and overexpression of wild-type epsinR impairs STxB/STx1B transport [30]. Additional factors that regulate adaptor recruitment also contribute to the retrograde transport of STxB/STx1B. One example is OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase that is associated with clathrin-coated, Golgi-directed intermediates [52; 32]. OCRL1 catalyzes the production of phosphatidylinositol 4-phosphate on early endosomal membranes [32], thus modulating adaptor recruitment. Additionally, OCRL1 directly interacts with clathrin and promotes clathrin assembly in vitro [32]. Overexpression of OCRL1 blocks early endosome-to-Golgi transport of STxB/STx1B [32]. Retromer is a heteropentameric coat complex composed of a sorting nexin (SNX) dimer and a Vps26-Vps29-Vps35 heterotrimer, which is also known as the cargo recognition complex [53]. SNX proteins have PX domains that interact with endosomal phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, and BAR domains that sense and/or generate membrane curvature [53]. siRNA-mediated silencing of SNX1 or SNX2 leads to a significant reduction in the early endosome-to-Golgi transport of STxB/STx1B [33; 34]. Furthermore, retrograde transport of STxB/STx1B is also impaired in Vps26 siRNA-treated cells [35]. Thus, retromer plays a critical role in STxB/STx1B transport. EHD3 and its early endosomal interaction partner rabenosyn-5 regulate recruitment of retromer to early endosomes and are also required for STxB/STx1B transport [36]. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Factor** & **Function** & **Reference** \\ \hline GPP130 & Endosomal sorting receptor & [19] \\ Cholesterol & Microdomain organization & [28] \\ Clathrin & Coat & [30; 31] \\ EpsinR & Clathrin adaptor & [30] \\ OCRL1 & Phosphatidylinositol 4,5-bisphosphate & [32] \\ Retromer (SNX1, SNX2, Vps26/29/35) & 5-phosphate & [33; 34; 35] \\ EHD3 & Retromer interactor & [36] \\ Rabenosyn5 & EHD35 interactor & [36] \\ RME-8 & Recruit Hesc-70 to SNX1 & [37] \\ Hsc-70 & Clathrin uncoating ATPase & [37] \\ Hrs & Clathrin interactor & [37] \\ Dynamin & Membrane scission & [31] \\ Microtubule & Cytoskeleton & [38] \\ Dynein & Motor & [38] \\ Actin & Cytoskeleton & [39] \\ Cdc42 & Rho GTPase & [39] \\ ARHGAP21 & Cdc12 activating protein & [39] \\ Rab11 & Small GTBase & [40; 41] \\ Rabe8  & Small GTBase & [41; 42] \\ Ar1 & Small GTBase, Golgi anchor & [43] \\ Golgi 97 & Tether, Ar11 effector & [43] \\ Golgi 245 & Tether, Ar11 effector & [44] \\ GCC185 & Tether, Ar11 effector & [45] \\ TMF & Tether, Rab6 effector & [46] \\ GARP & Tether & [47] \\ Syntaxia 5, Y4k6, GS15, GS28 & SNARE complex & [48] \\ Syntax 6, syntaxin 16, Vi1a, Vamp3/4 & SNARE complex & [41] \\ p38 & Kinase & [49] \\ PKC5 & Kinase & [50] \\ V-ATPase & Proton pump & [51] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Factors required for early endosome-to-Golgi transport of STxB/STx1B. Clathrin and retromer coats are believed to function in sequential transport steps, rather than in parallel, since disruption of either causes a nearly complete block in retrograde transport of STxB/STx1B [30; 35]. In clathrin-depleted cells, STxB/STx1B colocalizes with transferrin receptor, a canonical marker for early endosomes, while, in Vps26-depleted cells, STxB/STx1B is found in transferrin receptor-free endosomal tubules [30; 35].