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.

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 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...

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.

We employed electron cryo‐tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation‐arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.

Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.DOI: http://dx.doi.org/10.7554/eLife.07380.001

SummaryThe Sec complex forms the core of a conserved machinery coordinating the passage of proteins across or into biological membranes. The bacterial complex SecYEG interacts with the ATPase SecA or translating ribosomes to translocate secretory and membrane proteins accordingly. A truncated preprotein competes with the physiological full-length substrate and primes the protein-channel complex for transport. We have employed electron cryomicroscopy of two-dimensional crystals to determine the structure of the complex unlocked by the preprotein. Its visualization in the native environment of the membrane preserves the active arrangement of SecYEG dimers, in which only one of the two channels is occupied by the polypeptide substrate. The signal sequence could be identified along with the corresponding conformational changes in SecY, including relocation of transmembrane segments 2b and 7 as well as the plug, which presumably then promote channel opening. Therefore, we propose that the structure describes the translocon unlocked by preprotein and poised for protein translocation.

Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.