Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis

Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe(228) and Gly(230)) that are predicted to lie in the central pore region of the hexamer. The functions of Phe(228) and Gly(230) were probed by site-directed mutagenesis. The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228. None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade sigma(32), a physiological substrate. The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not. Mutation of these two pore residues also affected the ATPase activity of FtsH. The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity. The F228E mutant was actually more active ATPase. The substrates, sigma(32) and casein, stimulated the ATPase activity of wild type FtsH. The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe(228) mutants, but not the G230A mutant, remained sigma(32)-stimulatable. These results suggest that Phe(228) and Gly(230) in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis.