Abstract
AbstractThe 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. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals 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 thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.
Funder
U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences
Division of Intramural Research, National Institute of Allergy and Infectious Diseases
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry
Cited by
29 articles.
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