The p21 Rho-activating toxin cytotoxic necrotizing factor 1 is endocytosedby a clathrin-independent mechanism and enters the cytosol by an acidic-dependent membrane translocation step

Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic str ains of Escherichia coli, activates the p21 Rho-GTP-binding protein, induci ng a profound reorganization of the actin cytoskeleton. CNF1 binds to its c ell surface receptor on HEp-2 cells with high affinity (K-d = 20 pM). In HE p-2 cells the action of CNF1 is not blocked in the presence of filipin, a d rug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and interna lization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is simil ar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cro ss the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that require d by diyhtheria toxin. As shown for diphtheria toxin, the pH-dependent memb rane translocation step of CNF1 could be mimicked at the level of the plasm a membrane by a brief exposure to a FH of less than or equal to 5.2. CNF1 i s the first bacterial toxin described that uses both a clathrin-independent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol.