Figure 6.

CT Y149A and CT Y149S mutants are not activated by ARF6 and lack cytopathic activity.

A. Wild-type and mutant CT holotoxins were mixed with 2 mM DEA-BAG in the absence (grey bars) or presence (black bars) of ARF6/GTP for 2 h at 30°C. The ADP-ribosylation of DEA-BAG was then assessed by fluorometry; increasing fluorescent units correspond to increasing levels of ADP ribosylation. Background-subtracted data are presented as the averages ± ranges of two replicate samples per condition. One of three representative experiments is shown.

B. CHO cells were challenged with the stated concentrations of wild-type CT (filled circles), CT Y149A (open circles), or CT Y149S (open squares). Cell extracts generated after 2 h of continual toxin exposure were then screened for cAMP content. Data are presented as the means ± SEMs of 4 independent experiments with triplicate samples.

C. CHO cells pulse-labeled at 4°C with wild-type or mutant CT were subsequently chased at 37°C in toxin-free medium for 2 h. The cytosolic fractions extracted from intoxicated cells were perfused over a SPR sensor coated with an anti-CTA1 antibody. Unintoxicated cells and cells exposed to wild-type CT in the presence of brefeldin A (BfA), a drug that prevents toxin delivery to the cytosol, were used as negative controls. Known quantities of CTA (0.1 and 1 ng/mL) were used as positive controls. One of three representative experiments is shown.