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Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

Summary The Sec translocon performs protein secretion and membrane protein insertion at the plasma membrane of bacteria and archaea (SecYEG/β), and the endoplasmic reticular membrane of eukaryotes (Sec61). Despite numerous structures of the complex, the mechanism underlying translocation of pre-proteins, driven by the ATPase SecA in bacteria, remains unresolved. Here we present a series of biochemical and computational analyses exploring the consequences of signal sequence binding to SecYEG. The data demonstrate that a signal sequence-induced movement of transmembrane helix 7 unlocks the translocon and that this conformational change is communicated to the cytoplasmic faces of SecY and SecE, involved in SecA binding. Our findings progress the current understanding of the dynamic action of the translocon during the translocation initiation process. The results suggest that the converging effects of the signal sequence and SecA at the cytoplasmic face of SecYEG are decisive for the intercalation and translocation of pre-protein through the SecY channel., Graphical Abstract, Highlights • Validation of previously observed signal sequence-induced “unlocking” of SecYEG • Conformational changes upon SecYEG unlocking are relayed to SecA binding site • Unlocking the translocon perturbs the interaction between SecY and SecE • Conformational changes distinct between secretion and membrane protein insertion, Corey et al. have used biochemical and computational techniques to analyze conformational changes in the bacterial SecYEG translocon upon signal sequence binding. These structural effects are likely key to understanding how the translocon is primed for translocation by the concerted action of the signal sequence and the ATPase SecA.

Membrane proteins constitute about one third of the proteome. The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane. In Escherichia coli post- and co-translational targeting pathways converge at the protein-conducting channel, consisting of a central pore, SecYEG, which can recruit accessory domains SecDF-YajC and YidC, to form the holotranslocon (HTL) supercomplex. Detailed analysis of HTL function and architecture remained elusive until recently, largely due to the lack of a purified, recombinant complex. ACEMBL is an advanced DNA recombineering-based expression vector system we developed for producing challenging multiprotein complexes. ACEMBL affords the means to combine multiple expression elements including promoter DNAs, tags, genes of interest, and terminators in a combinatorial manner until optimal multigene expression plasmids are constructed that yield correctly assembled, homogenous, and active multiprotein complex specimens. We utilized ACEMBL for recombinant HTL overproduction. We developed protocols for detergent solubilizing and purifying the HTL. Highly purified complex was then used to reveal HTL function and the interactions between its constituents. HTL activity in protein secretion and membrane protein insertion was analyzed in both the presence and absence of the proton-motive force. Setting up ACEMBL for the assembly of multigene expression constructs that achieve high yields of functional multisubunit membrane protein complex is straightforward. Here, we used ACEMBL for obtaining active HTL supercomplex in high quality and quantity. The concept can likewise be applied to obtain many other assemblies of similar complexity, by overexpression in prokaryotic, and also eukaryotic hosts.

Membrane proteins constitute about one third of the proteome. The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane. In Escherichia coli post- and co-translational targeting pathways converge at the protein-conducting channel, consisting of a central pore, SecYEG, which can recruit accessory domains SecDF-YajC and YidC, to form the holotranslocon (HTL) supercomplex. Detailed analysis of HTL function and architecture remained elusive until recently, largely due to the lack of a purified, recombinant complex. ACEMBL is an advanced DNA recombineering-based expression vector system we developed for producing challenging multiprotein complexes. ACEMBL affords the means to combine multiple expression elements including promoter DNAs, tags, genes of interest, and terminators in a combinatorial manner until optimal multigene expression plasmids are constructed that yield correctly assembled, homogenous, and active multiprotein complex specimens. We utilized ACEMBL for recombinant HTL overproduction. We developed protocols for detergent solubilizing and purifying the HTL. Highly purified complex was then used to reveal HTL function and the interactions between its constituents. HTL activity in protein secretion and membrane protein insertion was analyzed in both the presence and absence of the proton-motive force. Setting up ACEMBL for the assembly of multigene expression constructs that achieve high yields of functional multisubunit membrane protein complex is straightforward. Here, we used ACEMBL for obtaining active HTL supercomplex in high quality and quantity. The concept can likewise be applied to obtain many other assemblies of similar complexity, by overexpression in prokaryotic, and also eukaryotic hosts.

The SecY/61 complex forms the protein-channel component of the ubiquitous protein secretion and membrane protein insertion apparatus. The bacterial version SecYEG interacts with the highly conserved YidC and SecDF-YajC subcomplex, which facilitates translocation into and across the membrane. Together, they form the holo-translocon (HTL), which we have successfully overexpressed and purified. In contrast to the homo-dimeric SecYEG, the HTL is a hetero-dimer composed of single copies of SecYEG and SecDF-YajC-YidC. The activities of the HTL differ from the archetypal SecYEG complex. It is more effective in cotranslational insertion of membrane proteins and the posttranslational secretion of a β-barreled outer-membrane protein driven by SecA and ATP becomes much more dependent on the proton-motive force. The activity of the translocating copy of SecYEG may therefore be modulated by association with different accessory subcomplexes: SecYEG (forming SecYEG dimers) or SecDF-YajC-YidC (forming the HTL). This versatility may provide a means to refine the secretion and insertion capabilities according to the substrate. A similar modularity may also be exploited for the translocation or insertion of a wide range of substrates across and into the endoplasmic reticular and mitochondrial membranes of eukaryotes.