Trafficking of cholera toxin-ganglioside GM1 complex into Golgi and induction of toxicity depend on actin cytoskeleton.
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.
Department of Pathology, Massachusetts General Hospital, Boston 02114, USA. kbadizadegan@partners.org
Kamran
K
Badizadegan
Heidi E
HE
Wheeler
Yukako
Y
Fujinaga
Wayne I
WI
Lencer
D000199:Actins / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000502:physiology; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D011233:Precipitin Tests; D021381:Protein Transport / Q000502:physiology*
D000199:Actins; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases