Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

Abstract

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.

MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

“…The machineries that insert and remove membrane proteins into and from the ER, respectively, induce a local membrane thinning, presumably to lower the energy barrier for insertion and extraction (Wu and Rapoport, 2021 ). Consequently, aberrantly high sterol levels or increased lipid saturation inhibit the insertion of transmembrane proteins in model membranes, the mammalian ER, and bacterial membranes (Nilsson et al, 2001 ; Brambillasca et al, 2005 ; Kamel et al, 2022 ). Therefore, it comes as no surprise that the compressibility and thickness of the ER membrane is continuously monitored by the UPR for regulating the relative rate of protein and lipid biosynthesis (Halbleib et al, 2017 ; Schuck et al, 2009 ; Covino et al, 2018 ).…”

A unifying mechanism for protein transport through the core bacterial Sec machinery

“…Beyond Sec-associated proteins and the PMF, the presence of specific lipids-notably cardiolipin (CL)-are important for ensuring that Sec transport is at its fastest [140][141][142]. Indeed, CL seems to be important for the conferring PMF-stimulation to transport across the inner membrane [140], perhaps by assisting in the rapid dissipation of protons away from translocating lysines at the cytosolic surface.…”

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors

“…Figure 6), thus pointing to intermolecular FRET at elevated protein-to-lipid ratios, either due to random contacts or due to clustering of the sensors in the lipid membrane. Based on those insights and the optimal signal-to-noise level, further experiments were conducted at the reconstitution ratio of 1:3,000, where a single sensor molecule would occur on average over 1,000 nm 2 area of the lipid bilayer (Hills & McGlinchey, 2016;Kamel et al, 2022).…”

“…Addition of Strep D4 did not influence the sensor conformation, as the FA/FDratios were not affected (1.15 ± 0.01 for αH-SecE and 2.21 ± 0.01 for (GSG)6-SecE; Figure 7D), the protein was not expected to interact with the membrane surface. To induce the interfacial crowding, we employed the ATPase SecA, as the protein contains an amphipathic N-terminal helix essential for docking SecA at the membrane interface (Kamel et al, 2022).…”

“…SecA N and SecA C (Kamel et al, 2022). As mSA was expressed as inclusion bodies and had to be refolded, its functionality was additionally analyzed by differential scanning fluorimetry (nanoDSF, Prometheus NT48).…”