FIG. 2.

Appearance of a 55-kDa fragment of CNF1 with deamidase activity. (A) HeLa cells were incubated with CNF1 or CNF1(E382/383K) (400 ng/ml each) for 4 h at 37°C in the absence or presence of bafilomycin A1 (Baf) and subsequently lysed. After fractionation of the lysates, the cytosol of toxin-treated cells and the cytosol of untreated control cells were separated by SDS-PAGE, and Western blotting with a monoclonal antibody against CNF1 was performed. A toxin fragment with a molecular mass of about 55 kDa was detected by the antibody only in the cytosol of CNF1-treated cells without bafilomycin A1. The CNF1 fragment has deamidase activity (B). HeLa lysates containing recombinant His-RhoA were incubated with the purified cytosols described above at 37°C overnight. Deamidation of His-RhoA was detected by the shift of the GTPase in SDS-PAGE in a Western blot against the His tag. Only cytosol which contained the CNF1 fragment had deamidase activity. The experiments were repeated more than three times with comparable results. ctrl, control; wt, wild type.