ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.

CIostridium perfiingens iota toxin ADP-ribosylates actin. Substrates of C. perfringens toxin are both nonmuscle /I/y-a&in and skeletal muscle actin. This finding suggests that C. perfringens iota ADP-ribosylates the same amino acid in skeletal muscle and non-muscle actin as does C. botulinwn C2 toxin in non-muscle actin. Protein chemical analysis involving thermolysin cleavage on [32P]ADP-ribosylated actin or tryptic digestion followed by a secondary thermolysin cleavage of the radiolabelled fragments showed one major site of ADP-ribosylation. From its amino acid composition and sequence, the radiolabelled peptide was identified as peptide 175-177, locating the acceptor ADP-ribosyl amino acid as Arg-177.

We studied the formation of actin scaffolds in giant vesicles of dimyristoylphosphatidylcholine (DMPC). Polymerization of actin was induced at low ionic strength through ionophore-mediated influx of Mg2+ (2 mM). The spatial organization of the filamentous actin was visualized by confocal and epifluorescence microscopy as a function of the filaments length and membrane composition, by including various amounts of cholesterol or lipids with neutral and positively charged polyethyleneglycol headgroups (PEG lipopolymers). In vesicles of pure DMPC, the newly polymerized actin adsorbs to the membrane and forms a thin shell. In the presence of 2.5 mol% lipopolymers or of cholesterol at a molar fraction x=0.37, formation of a thin adsorbed film is impeded. A fuzzy cortex is predominantly formed in vesicles of diameter d smaller than the filament persistence length (d< or =15 microm) while for larger vesicles a homogeneous network formation is favoured in the bulk of the vesicle. The fuzzy-cortex formation is interpreted as a consequence of the reduction of the bending energy if the actin filaments accumulate close to the vesicle wall.

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