Escherichia coli FtsH is an ATP-dependent and membrane-bound protease, which belongs to the ATPases associated with diverse cellular activities family. FtsH degrades a subset of cytoplasmic regulatory proteins and misassembled membrane proteins. It has been proposed that ATP-dependent proteases unfold and translocate substrate proteins into the protease chamber. Previously, we reported that Phe228 and Gly230 in the conserved motif, @XG (where @ is an aromatic residue and X is any residue), in the central pore of the FtsH ATPase ring have important roles in proteolysis and its coupling to ATP hydrolysis. In this paper, we constructed and characterized additional pore mutants. Results indicated that certain acidic residues located in the pore region are also important for the activity of FtsH. Proteolytic activities of most mutants are correlated with their ATPase activities. Evidence also indicated that Val229, the 2nd residue of the @XG motif, may have a substrate-specific role. (c) 2006 Elsevier Inc. All rights reserved.