Protein translocation: what's the problem?

Abstract

We came together in Leeds to commemorate and celebrate the life and achievements of Prof. Stephen Baldwin. For many years we, together with Sheena Radford and Roman Tuma (colleagues also of the University of Leeds), have worked together on the problem of protein translocation through the essential and ubiquitous Sec system. Inspired and helped by Steve we may finally be making progress. My seminar described our latest hypothesis for the molecular mechanism of protein translocation, supported by results collected in Bristol and Leeds on the tractable bacterial secretion process-commonly known as the Sec system; work that will be published elsewhere. Below is a description of the alternative and contested models for protein translocation that we all have been contemplating for many years. This review will consider their pros and cons.

Keywords: ATPase; SecY; energy transduction; membrane protein complex; protein translocation; secretion.

Figure 1. Structure of SecYEG–SecA

T. maritima SecYEG–SecA (PDB code 3DIN [29]). Proteins are represented as cartoons with mesh surfaces. SecY is light pink, with the partly opened LG helices highlighted in dark pink and the plug as grey. SecE is shown in orange, SecG in green and SecA in light-blue, with the 2HF and PPXD coloured separately. The ATP analogue (ADP-BeF_x) is coloured as orange, blue and red spheres. The approximate position of the membrane is indicated.

Figure 2. Modelling a pre-protein path through the complex

(A) Model built based on the SecYEG–SecA crystal structure (PDB code 3DIN [29]) which has been allowed to relax with MD simulations (full details to be published separately). The proteins are shown as cartoons, with SecA in light blue, SecY in pink, SecE in light orange and SecG in light green, with a bound ATP molecule shown as orange, blue, white and red spheres. A prospective pathway for a model substrate (the first 76 residues of pro-OmpA; shown as dark blue cartoon and mesh) has been built into the channel based on known cross-linking sites in SecA (pink, red, blue and white spheres; [35]) and the position of the SecY pore ring [30]. The helical SS was built based on the density of the DsbA SS from a recent cryo-EM structure of the SecY complex bound to a ribosome (inset–DsbA SS light blue, with the density shown using map EMD-5693, at 3.0 sigma within 2.6 Å of the selection [13]). (B) Close-up of the channel from (A) with the substrate, LG and 2HF shown as dark blue, pink and teal mesh respectively. The pore ring residues of SecY are highlighted in red.

Figure 3. Previously proposed models for translocation by the Sec complex

Many previous models for how SecA drives translocation have been proposed, designed to accommodate the results of structural and functional studies. So far, however, such models make various assumptions, e.g. they postulate the existence of conformational changes that lack direct experimental evidence. In general these can be divided into three types: models involving a power stroke within SecA, those that invoke quaternary interactions between multiple SecA molecules or those which act through biased diffusion. (A) An example power-stroke mechanism, whereby conformational changes within SecA during the ATPase cycle physically push polypeptides through the channel. In the model shown [36], the 2HF binds to the pre-protein substrate, pushes it into the channel, then releases it and returns to its resting position. (B) The observation that SecA can exist both as a monomer and in several different dimer forms has led to the proposal of multiple models in which quaternary interactions drive transport. In the example shown, one SecA protomer holds the pre-protein substrate in the channel whereas the other binds to downstream regions. ATP binding alters the SecA dimer interface, pushing the substrate through the channel, whereas ATP hydrolysis releases SecA, allowing it to rebind downstream. (C) Rather than physically pushing the substrate through the channel, directional movement can be achieved by selectively allowing diffusion in one direction, while preventing it in the other. Such a ‘Brownian ratchet’ would act by using ATP to somehow prevent backsliding. In the version shown, SecA senses backsliding and constricts to halt movement; however this is entirely speculative, as an illustration of the core concept.