Binding and internalization of Clostridium perfringens iota-toxin in lipid rafts

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). The oligomer of Ib formed in membranes induces endocytosis. We examined the binding and internalization of Ib by using Cy3-labeled Ib. Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37degreesC, and later it was detected in cytoplasmic vesicles. To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37degreesC and fractionated the Triton-insoluble membranes. An Ib complex of 500 kDa was localized at 37degreesC to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4degreesC. The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37degreesC. When the cells that were preincubated with Ib at 4degreesC were incubated at 37degreesC, the complex was detected in the raft fraction. The treatment of MDCK cells with methyl-p-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by la plus Ib. When 125 I-labeled la was incubated with the cells in the presence of Ib at 37degreesC, it was localized in the raft fraction. Surface plasmon resonance analysis revealed that la binds to the oligomer of Ib. We conclude that Ib binds to a receptor in membranes and then moves to rafts and that la bound to the oligomer of Ib formed in the rafts is internalized.