Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.
- MeSH
- adenosintrifosfát chemie metabolismus MeSH
- adenosintrifosfatasy chemie metabolismus MeSH
- Escherichia coli metabolismus MeSH
- membránové transportní proteiny chemie metabolismus MeSH
- molekulární modely MeSH
- proteinové prekurzory metabolismus MeSH
- proteiny SecA chemie metabolismus MeSH
- proteiny z Escherichia coli chemie metabolismus MeSH
- sbalování proteinů * MeSH
- translokační kanály SEC chemie metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- fluorescenční mikroskopie metody MeSH
- hydrolýza MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- mutace MeSH
- proteiny - lokalizační signály genetika MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- protonmotorická síla * MeSH
- translokační kanály SEC chemie genetika metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
