Abstract
The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins. SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein). We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH. We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini. Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH. Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. Mutant proteins without ATPase activity were inactive in proteolysis. A zinc binding motif mutant showed a decreased proteolytic activity. SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation. These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity. FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation.
Highlights
The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins
A1 site mutant proteins, FtsH40-His6-Myc (Fig. 4, lane 6) and FtsH41-His6Myc, did not appreciably degrade SecY. These results demonstrated that FtsH has an ATP-dependent protease activity toward the isolated SecY protein and that the ATPase region is important for the proteolytic activity
Proteolytic Activity of FtsH Is Selective—We examined how generally membrane proteins serve as a substrate for FtsH
Summary
The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins. Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity. An ATP-dependent protease activity of FtsH was described [11] It is involved in the degradation of an intrinsically unstable heat shock transcription factor, 32 [11, 13]. It was shown that FtsH homologs (Yta, Yta, and Osd1/Yme1) in the yeast mitochondria participate in degradation of some inner membrane proteins (14 –18) Their proteolytic activities have not been characterized in vitro using purified proteins. The results indicate that FtsH has an ATP-dependent protease activity with high substrate specificity
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