Individual SecYEG translocons visualized in supported lipid bilayers manifest dynamic heterogeneity in their lateral diffusion. Stable binding of ribosome :nascent chain complexes, but not SecA ATPase or empty ribosomes, severely hinders the translocon diffusion. The complex diffusion behaviour is attributed to the intrinsic translocon:lipid interactions and, potentially, the translocon conformational dynamics in the physiologically relevant environment.
The translocon SecYEG and the associated ATPase SecA form the primary protein secretion system in the cytoplasmic membrane of bacteria. The secretion is essentially dependent on the surrounding lipids, but the mechanistic understanding of their role in SecA : SecYEG activity is sparse. Here, we reveal that the unsaturated fatty acids (UFAs) of the membrane phospholipids, including tetraoleoyl-cardiolipin, stimulate SecA : SecYEGmediated protein translocation up to ten-fold. Biophysical analysis and molecular dynamics simulations show that UFAs increase the area per lipid and cause loose packing of lipid head groups, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced up to fivefold at elevated ionic strength. Tight SecA : lipid interactions convert into the augmented translocation. Our results identify the fatty acid structure as a notable factor in SecA : SecYEG activity, which may be crucial for protein secretion in bacteria, which actively change their membrane composition in response to their habitat.
Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and multiresistance against antibiotics. This bacterium produces and secretes various toxins with hydrolytic activities including phospholipases A, C and D. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. PaPlaB isolated from detergent-solubilized membranes of E. coli rapidly degraded various GPLs including endogenous GPLs isolated from P. aeruginosa cells. Cellular localization studies suggest that PaPlaB is peripherally bound to the inner and outer membrane of E. coli, yet the active form was predominantly associated with the cytoplasmic membrane. In vitro activity of purified and detergent-stabilized PaPlaB increases at lower protein concentrations. The size distribution profile of PaPlaB oligomers revealed that decreasing protein concentration triggers oligomer dissociation. These results indicate that homooligomerisation regulates PaPlaB activity by a yet unknown mechanism, which might be required for preventing bacteria from self-disrupting the membrane. We demonstrated that PaPlaB is an important determinant of the biofilm lifestyle of P. aeruginosa, as shown by biofilm quantification assay and confocal laser scanning microscopic analysis of biofilm architecture. This novel intracellular phospholipase B with a putative virulence role contributes to our understanding of membrane GPL degrading enzymes and may provide a target for new therapeutics against P. aeruginosa biofilms.
The translocon SecYEG forms the primary protein-conducting channel in the cytoplasmic membrane of bacteria, and the associated ATPase SecA provides the energy for the transport of secretory and cell envelope protein precursors. The translocation requires negative charge at the lipid membrane surface, but its dependence on the properties of the membrane hydrophobic core is not known. Here, we demonstrate that SecA:SecYEG-mediated protein transport is immensely stimulated by unsaturated fatty acids (UFAs). Furthermore, UFA-rich tetraoleoyl-cardiolipin, but not bis(palmitoyloleoyl)-cardiolipin, facilitate the translocation via the monomeric translocon. Biophysical analysis and molecular dynamics simulations show that UFAs determine the loosely packed membrane interface, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced manifold at elevated ionic strength. Tight SecA:lipid interactions convert into the augmented translocation. As bacterial cells actively change their membrane composition in response to their habitat, the modulation of SecA:SecYEG activity via the fatty acids may be crucial for protein secretion over a broad range of environmental conditions.
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