Stabilization of FtsH-unfolded protein complex by binding of ATP and blocking of protease

The function of an ATP-dependent membrane protease FtsH was investigated using the enzyme from Thermus thermophilus HB8. An FtsH mutant with replacement of Glu-419 in the zinc-binding motif by Cys lost the activity to digest casein, a model unfolded protein, and the small ATPase activity of this mutant was no longer stimulated by casein. In the presence of ATP or ATPgammaS, but not ADP, a mutant FtsH-unfolded protein complex was isolated, indicating that ATP binding, but not ATP hydrolysis, is required for FtsH to form a stable complex with an unfolded protein. The FtsH without mutation at Glu-419 did not produce a stable complex with casein in the presence of any nucleotides tested and therefore it appears that blocking proteolysis also contributes to stabilization of the complex.