AAA+ proteases are conserved molecular machines for degrading misfolded or superfluous proteins in cells. They utilize the free energy released from ATP hydrolysis to actively unfold a substrate protein on the ring AAA+-ATPase and translocate the unfolded protein to a protease chamber for proteolysis. Among bacterial AAA+ proteases, FtsH is only membrane-integrated and plays a pivotal role in quality control of membrane proteins. Previous studies using water-soluble globular proteins as model substrates have shown that FtsH has weak ATPase and unfoldase activities. This observation led to the proposal that FtsH cannot actively unfold a protein, and ATP hydrolysis is used only for substrate translocation, which confer the protease with an ability to selectively degrade intrinsically unstable or misfolded proteins. However, more recent studies indicate that FtsH can carry out the highly thermodynamically unfavorable processes, denaturation and membrane dislocation of stable membrane protein substrates by cooperatively utilizing ATP hydrolysis. These contradictory results provided a motivation for this study. Here, I clarified the role of the transmembrane (TM) domain of FtsH, that is, whether the TM domain acts simply as a membrane anchor or plays a role in unfolding and recognition of membrane proteins. By comparing, ATPase and unfoldase activities of a series of FtsH variants with modified TM domains against various substrates including both water-soluble and membrane proteins. I find a moderate inhibitory role of the native TM domain in hydrolyzing ATP and unfolding substrates. Surprisingly, FtsH can be converted into a highly efficient ATPase/unfoldase by modifying the TM domain. I also find that being integrated to the membrane does not hinder FtsH’s ability to unfold stable proteins, and this ATP-induced unfolding does not productively lead to degradation. Thus, substrate unfolding and translocation are not necessarily coupled during degradation by FtsH. Our results provide a new insight into energy-dependent protein degradation, that is, each AAA+ protease has a unique mechanism for degrading substrates.
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