The binary actin–ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.

The role of the cytoskeleton in the activity of GABA(A) receptors was investigated in cultured hippocampal neurons. Receptor currents were measured with the whole-cell patch-clamp technique during repetitive stimulation with 1 microm muscimol. After destruction of the microtubular system with nocodazol, muscimol-induced currents showed a rundown by 78%. A similar rundown was observed when actin fibers were destroyed with latrunculin B or C2 toxin of Clostridium botulinum. Because the small GTPases of the Rho family RhoA, Rac1, and Cdc42 are known to control the organization of actin fibers, we investigated their possible involvement. Inactivation of the GTPases with clostridial toxins, as well as intracellular application of recombinant Rho GTPases, indicated that active Rac1 was necessary for full GABA(A) receptor activity. Immunocytochemical labeling of the receptors showed that the disappearance of receptor clusters in the somatic membrane as induced by muscimol stimulation was enhanced by Rac1 inactivation. It is suggested that Rac1 participates in the regulation of GABA(A) receptor clustering and/or recycling.

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Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln 63 , caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30 -60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.The low molecular mass GTPases of the Rho family (Rho, Rac, and Cdc42) play key roles in the control of the actin cytoskeleton (1). In several cell lines, activation of RhoA induces formation of stress fibers and focal adhesions (2), whereas Rac and Cdc42 cause the formation of lamellipodia and filopodia, respectively (2-4). Moreover, Rho GTPases act as molecular switches in various signaling processes (5) including secretion (6), phagocytosis (7), endocytosis (8, 9), cell cycle progression (10), transcriptional activation (11) (22). C3 exoenzyme in particular is widely used as a cell biological tool to study the functions of Rho (23).Inasmuch as C3 exoenzyme appears to enter cells via nonspecific pinocytosis, its cell accessibility is poor (24). Recently, we constructed a C3 fusion toxin, which enters cells by using the binary C. botulinum C2 toxin as a carrier (25). The actin-ADP-ribosylating C2 toxin consists of the ϳ80-kDa binding component (C2II) and the 49-kDa enzymatic component (C2I) (26 -28). Both C2 components are separate proteins which assemble at the cell surface after C2II has bound to an unknown receptor. The C3 fusion toxin (C2IN-C3) contains the N-terminal domain (amino acid residues 1-225) of C2I (C2IN), which has no enzyme activity but binds to the C2II component and is sufficient for translocation of C2I into the cytosol. In contrast to C3 exoenzyme, the C2IN-C3 fusion toxin enters cells readily and causes the depolymerization of stress fibers at lo...

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