Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers

What is it about?

Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

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Why is it important?

We report here the visualization of translocation machinery at work in near-native lipid bilayers. Our comparison of translocases engaging two different precursors side by side offers a direct view of this complex and dynamic process. We imaged translocases at work under near-native conditions. AFM data complement high-resolution structures by providing a direct view of the Sec translocase engaging precursor proteins in a lipid bilayer and in aqueous buffer solution. Experiments showed translocase complexes undergoing significant topographic changes in a manner that depended on precursor species, nucleotide species, and stage of translocation

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