Dynamic action of the Sec machinery during initiation, protein translocation and termination.

Tomas Fessl,Daniel Watkins,Jim Horne,Roman Tuma,Steve A Baldwin,Peter Oatley,Ian Collinson,Robin Adam Corey,William John Allen,Sheena E Radford

doi:10.7554/elife.35112

Tomas Fessl, Daniel Watkins + Show 8 more

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https://doi.org/10.7554/elife.35112

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Journal: eLife	Publication Date: Jun 7, 2018
Citations: 57	License type: CC BY 4.0

Affiliation: University of Leeds, University of Bristol

Abstract

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.

Highlights

Protein secretion is essential for life; responsible for the delivery of proteins to and across the cell surface
Recent studies favour at least some element of diffusion: previously, we proposed a ‘pure’ ratchet model, in which the free energy available from ATP binding and hydrolysis at SecA drives a Brownian ratchet at the SecY Lateral Gate (LG) (Allen et al, 2016), while others have suggested a hybrid power stroke/diffusion model, in which ATP hydrolysis generates a power stroke (‘push’) on the polypeptide, followed by diffusion through the pore (‘slide’) (Bauer et al, 2014)
The plug helix is expected to relocate during activation of the channel by association of the Signal Sequence (SS) and SecA, and remains open during the protein translocation process (Bieker et al, 1990; Flower et al, 1995; Hizlan et al, 2012; Li et al, 2016; Robson et al, 2009a; Tam et al, 2005; Zimmer et al, 2008)

Summary

Introduction

Protein secretion is essential for life; responsible for the delivery of proteins to and across the cell surface. Pre-proteins are targeted to the Sec machinery with the aid of an N-terminal Signal Sequence (SS) or a Trans Membrane Helix (TMH), and translocated through the Sec machinery in an unfolded 41 conformation (Arkowitz et al, 1993) This can occur either during their synthesis (co translationally), or afterwards (post-translationally); in the latter case, pre-proteins are prevented from folding by cytosolic chaperones, such as SecB in bacteria (Kumamoto and Beckwith, 1983; Weiss et al, 1988). When at rest the channel is kept closed by a short, usually -helical plug and a ring of six hydrophobic residues, which serves to prevent ion leakage and dissipation of the Proton Motive Force (PMF) (Figure 1A) (Saparov et al, 2007) Separation of these domains opens a channel across the membrane (secretion) as well as a Lateral Gate (LG) for SS docking and membrane protein insertion (Figure 1B). Activation is achieved by the ribosome nascent chain complex (Jomaa et al, 2016; Voorhees and Hegde, 2016), or by association of pre protein and SecA (Corey et al, 2016b; Lill et al, 1989)

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