The SecA motor generates mechanical force during protein translocation

Abstract

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Monitoring translocation of a folded substrate protein with tunable stability at high time resolution allowed us to kinetically dissect Secdependent translocation. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we have also defined the response of the protein to mechanical force. Relating the kinetic and force measurements revealed that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements has thus allowed us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.