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

Abstract

Encapsulation and compartmentalization are fundamental to the evolution of cellular life, but they also pose a challenge: how to partition the molecules that perform biological functions-the proteins-across impermeable barriers into sub-cellular organelles, and to the outside. The solution lies in the evolution of specialized machines, translocons, found in every biological membrane, which act both as gate and gatekeeper across and into membrane bilayers. Understanding how these translocons operate at the molecular level has been a long-standing ambition of cell biology, and one that is approaching its denouement; particularly in the case of the ubiquitous Sec system. In this review, we highlight the fruits of recent game-changing technical innovations in structural biology, biophysics and biochemistry to present a largely complete mechanism for the bacterial version of the core Sec machinery. We discuss the merits of our model over alternative proposals and identify the remaining open questions. The template laid out by the study of the Sec system will be of immense value for probing the many other translocons found in diverse biological membranes, towards the ultimate goal of altering or impeding their functions for pharmaceutical or biotechnological purposes.

Keywords: Sec machinery; SecA; SecYEG; bacterial secretion; protein transport.

Figure 1.

Structures of SecA and SecYEG. (a) The first structure of SecA alone (from Bacillus subtilis; PDB code 1M6N [48]). Domains and key features are demarcated by colour as indicated (see also text for details), and movements of the PBD and NBD2 in response to SecYEG/pre-protein binding are indicated by dotted arrows. (b) The first structure of SecYEG (from Methanococcus jannaschii; PDB code 1RHZ [49]). Key features are labelled and distinguished by colour as noted.

Figure 2.

Initiation of protein translocation and pre-protein interactions. (a) Surface representation of SecYE-SecA with bound pre-protein before initiation (model based on PDB structure 6ITC [66,67]), coloured as in figure 1. The PBD of SecA has been removed and the C-terminal half of SecY made translucent (with the backbone in ribbon representation), to reveal the inner workings of the complex (compete structure shown as inset). (b) Closeup of the SecA clamp; see text for further details. (c) Cytosol-facing surface of SecA, coloured by hydrophobicity (red is more hydrophobic), as per [68]. Dashed circles indicate hydrophobic interaction sites for upstream mature regions of the translocating pre-protein [60]. (d) As in panel a, but post-initiation (PDB code 6ITC [66]). (e) Closeup of the post-initiation LG, coloured by hydrophobicity.

Figure 3.

Mechanism of transport. (a) Overview of the entire transport process for a pre-protein transported in multiple STEPS (5 as an example; see text for more detail). To reduce figure complexity, the Sec machinery is not shown in later STEPS. The black box highlights a single transport STEP, with proposed mechanisms expanded upon in panels (b–d). (b) Proposed helicase-like mechanism for ATP-driven transport by SecA, where alternate binding of TCβ and the PP-loop drives transport (see text) [97]. (c) Proposed ATP-driven mechanism of transport based on a power stroke by the 2HF [103]. (d) Proposed unifying model of ATP-driven transport (detailed in the main text). The entire panel corresponds to a single transport STEP, and is thus one of many repeats (five in this example; orange arrows) to fully transport the protein depicted in panel (a). Forward diffusion (magenta box) can be further augmented by other factors, as in panels (e–g). (e–g) Additional factors that promote forward or prevent reverse polypeptide diffusion during the ATPase cycle: (e) electrophoresis of negatively charged residues (Glu and Asp) promotes forward movement and inhibits its reverse; (f) ‘proton ratcheting’ of Lys (and potentially His) prevents backsliding; (g) regulated unfolding of pre-protein in the cytosol promotes forward movement (left), while refolding in the periplasm, binding of downstream periplasmic factors such as chaperones, or direct pulling as proposed for SecDF all inhibit reverse polypeptide movement, and thereby promote forward translocation (right).