Forces and Dynamics in Protein Translocation through the Bacterial Translocon

Many membrane and secretory proteins are translocated across the endoplasmic reticulum membrane or the bacterial/archaeal plasma membrane through a conserved channel, the Sec61 or the SecYEG complex, respectively. In post-translational translocation the SecYEG channel associates with the cytoplasmic motor protein SecA to deliver secretory proteins to the periplasm. In this study we characterized the binding of SecA to SecYEG at the single molecule level. For our AFM measurements, a sandwich structure was established that consisted of a mica-supported lipid bilayer containing biotinylated lipids, followed by a streptavidin layer, and SecYEG reconstituted into planar lipid bilayers, also containing biotinylated lipids. By covalently coupling SecA to AFM cantilevers we performed combined recognition imaging and force spectroscopy experiments. By varying the loading rate we obtained information on interaction forces, energy landscapes and kinetic rate-constants of the bond formed between SecA and SecYEG. No significant differences were observed in the presence and absence of ATP. In contrast, SecA only binds in the presence of ATP to SecYEG in detergent. This observation suggests that ATP and the lipid bilayer might induce similar conformational changes in SecA. However, ATP did not restore binding of a N-terminal deletion mutant of SecA to either pure lipid bilayers or reconstituted SecYEG, raising the possibility that SecYEG¯s binding interface adopts different conformations in detergent and the lipid environment. This work is supported by the Austrian Science Fund (FWF), no.W1250-B200.