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.

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951–962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (K d ≈ 5 nM), only CT elicited a cAMP-dependent Cl− secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.

To induce toxicity, cholera toxin (CT) must first bind ganglioside G M1 at the plasma membrane, enter the cell by endocytosis, and then traffic retrograde into the endoplasmic reticulum. We recently proposed that G M1 provides the sorting motif necessary for retrograde trafficking into the biosynthetic/secretory pathway of host cells, and that such trafficking depends on association with lipid rafts and lipid raft function. To test this idea, we examined whether CT action in human intestinal T84 cells depends on membrane cholesterol. Chelation of cholesterol with 2-hydroxypropyl ␤-cyclodextrin or methyl ␤-cyclodextrin reversibly inhibited CT-induced chloride secretion and prolonged the time required for CT to enter the cell and induce toxicity. These effects were specific to CT, as identical conditions did not alter the potency or toxicity of anthrax edema toxin that enters the cell by another mechanism. We found that endocytosis and trafficking of CT into the Golgi apparatus depended on membrane cholesterol. Cholesterol depletion also changed the density and specific protein content of CT-associated lipid raft fractions but did not entirely displace the CT-G M1 complex from these lipid raft microdomains. Taken together these data imply that cholesterol may function to couple the CT-G M1 complex with raft domains and with other membrane components of the lipid raft required for CT entry into the cell.Vibrio cholerae causes worldwide epidemics of life-threatening secretory diarrhea by colonizing the intestinal lumen and producing cholera toxin (CT), 1 a potent enterotoxin that invades the intestinal epithelial cell as a fully folded protein.Structurally, CT consists of two components. The pentameric B-subunit binds stoichiometrically to five G M1 gangliosides on the apical (lumenal) surface of intestinal epithelial cells, and the enzymatic A-subunit activates adenylyl cyclase inside the cell by catalyzing the ADP-ribosylation of the heterotrimeric GTPase G s (1). Activation of adenylyl cyclase in intestinal crypt epithelial cells leads to Cl Ϫ secretion, the fundamental transport event in secretory diarrhea.To induce disease, both A-and B-subunits must enter the host epithelial cell as a fully assembled holotoxin by moving retrograde through the biosynthetic pathway into the ER, 2 where the A-subunit unfolds, dissociates from the B-pentamer, and translocates to the cytosol, presumably by dislocation through the protein conducting channel sec61p (2). The time between CT binding to G M1 at the cell surface and induction of toxicity has been termed the "lag phase." The lag phase corresponds to the time required for trafficking CT into the ER, unfolding of the A-subunit by interaction with protein-disulfide isomerase, and finally dislocation of the A-subunit into the cytosol (2, 3).In the model polarized intestinal epithelial cell line T84, CT function depends on B-subunit binding to (and possibly clustering) ganglioside G M1 . Binding to G M1 anchors CT to the host cell membrane and associates CT with cholesterol-ri...