Single‐molecule analysis of dynamics and interactions of the SecYEG translocon
Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 mu m(2)center dot s(-1). Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.
|Cell Biology, Biochemistry, Molecular Biology|
Koch, S, Seinen, A.B, Kamel, M, Kuckla, D, Monzel, C, Kedrov, A, & Driessen, A.J.M. (2020). Single‐molecule analysis of dynamics and interactions of the SecYEG translocon. FEBS J.. doi:10.1111/febs.15596