Cholera toxin (CT) travels from the plasma membrane of intestinal cells to the endoplasmic reticulum (ER) where a portion of the A-subunit, the A1 chain, crosses the membrane into the cytosol to cause disease. A related toxin, LTIIb, binds to intestinal cells but does not cause toxicity. Here, we show that the B-subunit of CT serves as a carrier for the A-subunit to the ER where disassembly occurs. The B-subunit binds to gangliosides in lipid rafts and travels with the ganglioside to the ER. In many cells, LTIIb follows a similar pathway, but in human intestinal cells it binds to a ganglioside that fails to associate with lipid rafts and it is sorted away from the retrograde pathway to the ER. Our results explain why LTIIb does not cause disease in humans and suggest that gangliosides with high affinity for lipid rafts may provide a general vehicle for the transport of toxins to the ER.
Intestinal epithelial lipid rafts contain ganglioside GM1 that is the receptor for cholera toxin (CT). The ganglioside binds CT at the plasma membrane (PM) and carries the toxin through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER). In the ER, a portion of the toxin unfolds and translocates to the cytosol to activate adenylyl cyclase. Activation of the cyclase leads to an increase in intracellular cAMP, which results in apical chloride secretion. Here, we find that an intact actin cytoskeleton is necessary for the efficient transport of CT to the Golgi and for subsequent activation of adenylyl cyclase. CT bound to GM1 on the cell membrane fractionates with a heterogeneous population of lipid rafts, a portion of which is enriched in actin and other cytoskeletal proteins. In this actin-rich fraction of lipid rafts, CT and actin colocalize on the same membrane microdomains, suggesting a possible functional association. Depolymerization or stabilization of actin filaments interferes with transport of CT from the PM to the Golgi and reduces the levels of cAMP generated in the cytosol. Depletion of membrane cholesterol, which also inhibits CT trafficking to the TGN, causes displacement of actin from the lipid rafts while CT remains stably raft associated. On the basis of these observations, we propose that the CT-GM1 complex is associated with the actin cytoskeleton via the lipid rafts and that the actin cytoskeleton plays a role in trafficking of CT from the PM to the Golgi/ER and the subsequent activation of adenylyl cyclase.
In intestinal epithelia, cholera and related toxins elicit a cAMP-dependent chloride secretory response fundamental to the pathogenesis of toxigenic diarrhea. We recently proposed that specificity of cholera toxin (CT) action in model intestinal epithelia may depend on the toxin's cell surface receptor ganglioside G(M1). Binding G(M1) enabled the toxin to elicit a response, but forcing the toxin to enter the cell by binding the closely related ganglioside G(D1a) rendered the toxin inactive. The specificity of ganglioside function correlated with the ability of G(M1) to partition CT into detergent-insoluble glycosphingolipid-rich membranes (DIGs). To test the biological plausibility of these hypotheses, we examined native human intestinal epithelia. We show that human small intestinal epithelia contain DIGs that distinguish between toxin bound to G(M1) and G(D1a), thus providing a possible mechanism for enterotoxicity associated with CT. We find direct evidence for the presence of caveolin-1 in DIGs from human intestinal epithelia but find that these membranes are heterogeneous and that caveolin-1 is not a structural component of apical membrane DIGs that contain CT.
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