FIG. 5.

Identification of the cleavage site region. (A) Summary of changes in morphology (morphol.), in vitro fragment formation, and Rho shift after treatment of HeLa cells with CNF1 mutants in the putative cleavage site region. The results of at least three independent experiments are summarized. ++, the activity was the same as the activity with wild-type CNF1 for induction of the typical morphology (flattening, ruffling, formation of filopodia, polynucleation) or for the ability to deamidate RhoA (shift) or a comparable amount of the CNF1 fragment was detectable by Western blotting; +, less activity or less fragment than that observed for wild-type CNF1; −, no detectable morphological changes, no ability to shift RhoA, or no fragment in the cytosol. ctrl., control; wt, wild type. (B) Morphological changes in HeLa cells induced by CNF1 and CNF1 mutants. HeLa cells were treated with CNF1 or CNF1 mutants (400 ng/ml each) as indicated. The cells were intoxicated for 4 h, and cytosolic fractions were prepared. (C) C-terminal fragment as revealed by Western blot analysis against the catalytic domain of CNF1. α-CNF1, anti-CNF1. (D) For the in vitro Rho shift, cytosols were separated by urea-SDS-PAGE, and the shift was detected with a monoclonal antibody against RhoA (α-RhoA). (E) TER after CNF1 intoxication in Caco-2 cells grown on filters. Cells were treated with 100 ng/ml CNF1, inactive mutant CNF1(C866S), pore-forming deficient mutant CNF1(E383K/E384K), and two representative cleavage region mutants, CNF1(N534S/I535L) and CNF1(P536A/V537G). TER was measured after 4 h (open bars), 6 h (striped bars), and 8 h (filled bars). The results are expressed as the percentage of the TER at time zero, and the bars and error bars indicate the means ± standard deviations of at least three independent experiments. (F) Amino acid sequence of the cleavage site region of CNF1 (amino acids 526 to 548). Amino acids identified as amino acids necessary for CNF1 processing are indicated by larger type.