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

Abstract

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.

Methodologies for Measuring Protein Trafficking across Cellular Membranes

“…[77] By reconstituting individual translocons on glass-or mica-supported lipid bilayers, King and colleagues directly measured SecYEG-SecA dynamics during translocation of model peptide substrates proOmpA and proGBP (Figure 5C). [78,79] They confirmed expected heights for the translocon at distinct stages of translocation and SecA engagement and ATP consumption is similar on the surface to that observed in solution. The translocation rate is about 15fold (for glass) to 70-fold (for mica) lower than in solution, which they ascribe to the requirement that newly translocated peptides squeeze into the aqueous layer between the lipid bilayer and the mica or glass surface.…”

“…[74] C) Determining SecA binding dynamics by measuring the height of a mica surfacesupported translocon complex (SecYEG.SecA) by atomic force microscopy (AFM). [79] D) The translocon conformational distribution depends on substrate identity. PDB 6R7L [82] and 3DIN [83] are used to show SecYEG and SecYEG.SecA complexes, respectively.…”

“…Adv. 2019, 5, eaav9404 [79] © The Authors, some rights reserved; exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license http://creativecommons.org/licenses/by-nc/4.0/…”

Characterizing the Locus of a Peripheral Membrane Protein–Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli

“…The complexity of the general secretory system has obfuscated central facets, including the fundamental nature of the translocation mechanism. “Power-stroke”, “Brownian ratchet”, and other mechanisms have been proposed, ,,, and recent experiments have shown aspects of the process that vary with precursor species. − What is clear is that proteins are generated at bacterial ribosomes at a rate of ≈20 amino acids per second . Measured rates for SecA-driven translocation of the precursor of outer membrane protein A through SecYEG range from about 5 to 40 amino acids per second. − The mean bound-state lifetime from our dynamic force spectroscopy experiments of SecA2–11 and E.…”

Protein Translocation Activity in Surface-Supported Lipid Bilayers

“…16 AFM has been utilized to study Sec system components in isolation as well as in supported lipid bilayers. 7,10,12,15,17,18 The advantages which AFM provides include the ability to monitor conformational dynamics in solution without the need for additional labeling. However, the technique requires that the specimens be adsorbed to a supporting surface, though bilayer stacking, tethering, or other cushioning methods have been developed to modulate the submembrane space.…”