Single molecule studies of the E.coli protein export system (997.9)

Abstract

The general secretory or Sec system is the major route of export for proteins from the cytosol of Escherichia coli. The pathway through the membrane is provided by the translocon SecYEG. At the membrane, ATPase SecA binds SecYEG and drives protein translocation. In this work we shed light on Sec system using atomic force microscopy (AFM), a single molecule technique that is well suited for studying membrane proteins in near native conditions. AFM images provided direct visualizations of the dynamic loops of SecYEG which orchestrate critical translocon functions as well as determination of the oligomeric state of SecYEG in lipid bilayers. Additionally, we studied structure‐function relationships of SecA‐SecYEG complexes. Our results show that SecA binds SecYEG in liposomes in two distinct modes. When SecA was added extraneously to liposomes containing SecYEG, the proteins showed a wide distribution of heights with no prominent peaks, indicating that SecA populates multiple binding states. In contrast to this broad distribution of heights, the sample of proteoliposomes in which SecY and SecA were co‐assembled had a predominant species centered at 40 Å in height above the lipid bilayer. Because these proteoliposomes contain approximately sixfold more active translocons than do the proteoliposomes to which SecA was added after assembly, we conclude that this structural state represents SecA bound to translocons that have been rendered active. Taken together this work provides new functional and structural insights into the Sec system. More generally, the assays developed here are adaptable to other membrane protein systems.Grant Funding Source: NSF CAREER Award , Burroughs Wellcome Fund Career Award, NIH