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Protein and Vesicle Trafficking, Cytoskeleton

Trafficking of cholera toxin-ganglioside G_M1 complex into Golgi and induction of toxicity depend on actin cytoskeleton

¹Department of Pathology, Massachusetts General Hospital, and ²Department of Pathology, Harvard Medical School, Boston 02114; ³Gastrointestinal Cell Biology, Children's Hospital Boston, Boston, Massachusetts 02115; ⁴Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8530, Japan; and ⁵Department of Pediatrics, Harvard Medical School, and ⁶Harvard Digestive Diseases Center, Boston, Massachusetts 02115

Submitted 15 April 2004 ; accepted in final form 12 July 2004

Intestinal epithelial lipid rafts contain ganglioside G_M1 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 G_M1 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-G_M1 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.

membrane microdomains; membrane lipids; bacterial toxins; endocytosis; intestinal mucosa