Immobilization of the Plug Domain Inside the SecY Channel Allows Unrestricted Protein Translocation

The SecYEG complex forms a protein-conducting channel in the inner membrane of Escherichia coli to support the translocation of secretory proteins in their unfolded state. The SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. This helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10. Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggest that only a limited movement of the plug domain suffices for translocation.     

Deregulation of the SecYEG translocation channel upon removal of the plug domain

Previous studies have shown that the SecY plug is displaced from the center of the SecYEG channel during polypeptide translocation. The structural and functional consequences of the deletion of the plug are now examined. Both in vivo and in vitro observations indicate that the plug domain is not essential to the function of the translocon. In fact, deletion of the plug confers to the cell and to the membranes a Prl-like phenotype: reduced proton-motive force dependence of translocation, increased membrane insertion of SecA, diminished requirement for functional leader peptide, and weakened SecYEG subunit association. Although the plug domain does not seem essential, locking the plug in the center of the channel inactivates the translocon. Thus, the SecY plug is important to regulate the activity of the channel and to confer specificity to the translocation reaction. We propose that the plug contributes to the gating mechanism of the channel by maintaining the structure of the SecYEG complex in a compact closed state.     

Investigating the SecY plug movement at the SecYEG translocation channel

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.     

Conformational dynamics of the plug domain of the SecYEG protein-conducting channel

The central pore of the SecYEG preprotein-conducting channel is closed at the periplasmic face of the membrane by a plug domain. To study its conformational dynamics, the plug was labeled site-specifically with an environment-sensitive fluorophore. In the presence of a stable preprotein translocation inter-mediate, the SecY plug showed an enhanced solvent exposure consistent with a displacement from the hydrophobic central pore region. In contrast, binding and insertion of a ribosome-bound nascent membrane protein did not alter the plug conformation. These data indicate different plug dynamics depending on the ligand bound state of the SecYEG channel.     

Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the protein-conducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved.     

Escherichia coli Membranes Depleted of SecYEG Elicit SecA-Dependent Ion-Channel Activity but Lose Signal Peptide Specificity

We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.     

Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon

During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.     

Projection structure and oligomeric properties of a bacterial core protein translocase

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 protein-conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 A 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.     

Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.     

Synthetic peptides identify a second periplasmic site for the plug of the SecYEG protein translocation complex

A short helix in the centre of the SecY subunit serves as a ‘plug’ blocking the protein channel. This site must be vacated if the channel is to open and accommodate translocating protein. We have synthesised a peptide mimic of this plug, and show that it binds to E. coli SecYEG, identifying a distinct and peripheral binding site. We propose that during active translocation the plug moves to this second discrete site and chart its position. Deletion of the plug in SecY increases the stoichiometry of the peptide-SecYEG interaction by also exposing the location it occupies in the channel. Binding of the plug peptide to the channel is unaffected by SecA.     

Structure of the SecY complex unlocked by a preprotein mimic

The 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.     

The oligomeric state and arrangement of the active bacterial translocon

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.     

The protein-conducting channel SecYEG

In bacteria, the translocase mediates the translocation of proteins into or across the cytosolic membrane. It consists of a membrane embedded protein-conducting channel and a peripherally associated motor domain, the ATPase SecA. The channel is formed by SecYEG, a multimeric protein complex that assembles into oligomeric forms. The structure and subunit composition of this protein-conducting channel is evolutionary conserved and a similar system is found in the endoplasmic reticulum of eukaryotes and the cytoplasmic membrane of archaea. The ribosome and other membrane proteins can associate with the protein-conducting channel complex and affect its activity or functionality.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis.

In Escherichia coli, the SecYEG complex mediates the translocation and membrane integration of proteins. Both genetic and biochemical data indicate interactions of several transmembrane segments (TMSs) of SecY with SecE. By means of cysteine scanning mutagenesis, we have identified intermolecular sites of contact between TMS7 of SecY and TMS3 of SecE. The cross-linking of SecY to SecE demonstrates that these subunits are present in a one-to-one stoichiometry within the SecYEG complex. Sites in TMS3 of SecE involved in SecE dimerization are confined to a specific alpha-helical interface and occur in an oligomeric SecYEG complex. Although cross-linking reversibly inactivates translocation, the contact between TMS7 of SecY and TMS3 of SecE remains unaltered upon insertion of the preprotein into the translocation channel. These data support a model for an oligomeric translocation channel in which pairs of SecYEG complexes contact each other via SecE.     

The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure

Escherichia coli preprotein translocase comprises a membrane-embedded trimeric complex of SecY, SecE and SecG. Previous studies have shown that this complex forms ring-like assemblies, which are thought to represent the preprotein translocation channel across the membrane. We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross-linking and blue native gel electrophoresis. The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers. SecG-dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only. The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers. Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein. These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG.     

SecA alone can promote protein translocation and ion channel activity: SecYEG increases efficiency and signal peptide specificity

SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1-2 μM. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity.     

SecYEG assembles into a tetramer to form the active protein translocation channel

Translocase mediates preprotein translocation across the Escherichia coli inner membrane. It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA. Here we show by functional assays, negative-stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel. The active assembly of SecYEG has a side length of 10.5 nm and exhibits an approximately 5 nm central cavity. The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocase-precursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex

The bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA–SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation.