A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.

Transport and membrane integration of polypeptides is carried out by specific protein complexes in the membranes of all living cells. The Sec transport path provides an essential and ubiquitous route for protein translocation. In the bacterial cytoplasmic membrane, the channel is formed by oligomers of a heterotrimeric membrane protein complex consisting of subunits SecY, SecE and SecG. In the endoplasmic reticulum membrane, the channel is formed from the related Sec61 complex. Here we report the structure of the Escherichia coli SecYEG assembly at an in-plane resolution of 8 A. The three-dimensional map, calculated from two-dimensional SecYEG crystals, reveals a sandwich of two membranes interacting through the extensive cytoplasmic domains. Each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices. In the centre of the dimer we observe a 16 x 25 A cavity closed on the periplasmic side by two highly tilted transmembrane helices. This may represent the closed state of the protein-conducting channel.

Significance In this paper, we describe the biophysical properties, stoichiometry, and activity of the Escherichia coli SecYEG–SecDF–YajC–YidC holo-translocon. This multiprotein complex consists of seven membrane protein subunits, including those components responsible for both protein secretion (SecYEG) and membrane protein insertion (YidC). We demonstrate the isolation of a stable complex containing YidC together with the core SecY translocon. The availability of this intact assembly allows us to reconstitute posttranslational protein export and cotranslational membrane protein insertion from purified components of known stoichiometry. The experiments demonstrate that protein secretion and insertion occur through a single complex. The reconstitution of membrane protein insertion from defined components is a novel development, breaking ground for the functional analysis of this largely unknown process.

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.DOI: http://dx.doi.org/10.7554/eLife.15598.001

Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes.

The major route for protein export or membrane integration in bacteria occurs via the Sec-dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a proteinconducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, puri®ed and crystallized in two dimensions. A 9 A Ê projection structure was calculated using electron cryo-microscopy. The structure exhibits P12 1 symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.

The motor protein SecA drives the transport of polypeptides through the ubiquitous protein channel SecYEG. Changes in protein-nucleotide binding energy during the hydrolytic cycle of SecA must be harnessed to drive large conformational changes resulting in channel opening and vectorial substrate polypeptide transport. Here, we elucidate the ATP hydrolysis cycle of SecA from Escherichia coli by transient and steady-state methods. The basal ATPase activity of SecA is very slow with the release of ADP being some 600-fold slower than hydrolysis. Upon binding to SecYEG the release of ADP is stimulated but remains rate-limiting. ADP release is fastest in the fully coupled system when a substrate protein is being translocated; in this case hydrolysis and ADP release occur at approximately the same rate. The data imply that ADP dissociation from SecA is accompanied by a structural rearrangement that is strongly coupled to the protein interface and protein translocation through SecYEG.ATPase ͉ SecYEG ͉ steady-state kinetics ͉ transient kinetics ͉ translocon A TP-dependent molecular motors couple hydrolytic power to mechanical movement in a controlled and directional fashion. Distortions in the structure of the active site driven by different chemical stages in the ATP hydrolysis cycle can be relayed across large distances, resulting in conformational changes in the ATPase and its interacting partners. Hence, ATP binding, formation of the initial ADP:Pi complex or release of either of the products can be coupled to different motions required to drive the protein machine. The reverse can also be accomplished in the capture of free energy e.g., by the F 1 F 0 -ATP synthase (1, 2).The subject of this study, SecA, is an ATPase and the active component of the bacterial protein translocation machinery that also contains the protein channel SecYEG (3, 4). It peripherally associates with the membrane, receives unfolded secretory proteins and facilitates their passage across the inner membrane (5, 6). The structure of SecA reveals a single nucleotide binding site situated between 2 RecA-like folds, a scaffold, wing and preprotein cross-linking domain (7).Addition to SecA of preprotein and SecYEG reconstituted into an acidic phospholipid membrane promotes protein translocation and an increase in the rate of ATP hydrolysis (4,8). This corresponds to the energy-transducing activity and is a multistep process (9-11). How the structure of SecA or the channel (12, 13) relate to specific stages of the ATP coupled reaction is unclear.Although there is still controversy in the field as to the nature and purpose of homo-oligomerisation in SecA, an increasing body of evidence suggests that SecA is dimeric in free solution, but dissociates into monomers as a consequence of the protein transport reaction (14-21). Additional rearrangements within SecA (14,19,21,22) and SecYEG (21,23,24) have also been reported. These studies have been crowned by an X-ray structure of the complex (one copy of each) that reveals the nature and consequences of the...