Cholera toxin (CT), and members of the AB(5) family of toxins enter host cells and hijack the cell's endogenous pathways to induce toxicity. CT binds to a lipid receptor on the plasma membrane (PM), ganglioside GM1, which has the ability to associate with lipid rafts. The toxin can then enter the cell by various modes of receptor-mediated endocytosis and traffic in a retrograde manner from the PM to the Golgi and the endoplasmic reticulum (ER). Once in the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol so as to induce disease. GM1 is the vehicle that carries CT from PM to ER. Thus, the toxin pathway from PM to ER is a lipid-based sorting pathway, which is potentially meditated by the determinants of the GM1 ganglioside structure itself.

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endosomal sorting of the toxin-GM1 complex. IntroductionCholera toxin (CT) is an AB 5 -subunit toxin responsible for the massive secretory diarrhea seen in epidemic cholera. As for most toxins, CT must gain access to the cytosol of host cells to cause disease. The strategy employed by CT is to bind ganglioside GM1 in the plasma membrane (PM) via the B-subunit (CTB). GM1 carries the toxin retrograde through endosomes, the trans-Golgi network (TGN), and likely all the way into the ER (1, 2). In the ER, a portion of the A-subunit (CTA), the A1-chain, crosses to the cytosol by coopting the machinery that retro-translocates terminally misfolded proteins for degradation by the proteasome (termed ER-associated degradation [ERAD]; refs. 3, 4). The A1-chain refolds in the cytosol and activates adenylate cyclase to increase cAMP. The mechanisms for lipid sorting and ERAD usurped by CT are fundamental to eukaryotic cell biology but remain incompletely understood. To explore how CT exploits these pathways in an unbiased way, we used the zebrafish as a model because it is amenable to genetic screens. Here, we show that CT intoxicates zebrafish embryos by hijacking the same basic mechanisms used in mammalian cells and examine the dependence of CT toxicity on two families of proteins implicated in toxin action: the flotillins and Derlins. These proteins have emerged as important components of lipid-based trafficking and ERAD, respectively.There is evidence that GM1 sorts CT retrograde from PM to ER by association with lipid rafts (2, 5-8). Lipid rafts are cooperative selfassemblies of lipids and proteins that influence various aspects of

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