Dimeric SecA couples the preprotein translocation in an asymmetric manner

The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles.     

SecA: a tale of two protomers

Bacteria, Archaea and Eukaryotes have evolved a plethora of mechanisms to translocate proteins across their various membranes. The bacterial Sec pathway is ubiquitous and essential for cell viability and is used by most proteins destined for the inner membrane, the periplasm or beyond. In bacteria, Sec system components include the heterotrimers SecY/SecE/SecG and SecD/SecF/YajC and the peripherally associated ATPase motor SecA. SecA in solution is mainly dimeric. Unexpectedly, structures of SecA dimers from different or even the same bacterium do not have a consistent dimerization interface. Analysis of the functional assembled translocase complexes blurs the picture even further as the functional quaternary state of the SecYEG channel is also disputed. Several experimental approaches tried to define the oligomeric state of SecA during preprotein ‘pushing’ through SecYEG. One high‐resolution SecA–SecYEG complex has been visualized. This snapshot might be a step closer to the actual translocating machinery. Nevertheless, because of the use of detergent, the true quartenary state of the translocase might have been disturbed. Hence, even after this and other studies, several issues remain puzzling. New approaches must be combined with current tools to gain insight into the functionally relevant quartenary states of SecA and SecYEG during preprotein translocation.     

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.     

Two distinct anionic phospholipid-dependent events involved in SecA-mediated protein translocation

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.     

A single amino acid substitution in SecY stabilizes the interaction with SecA.

The SecYEG complex constitutes a protein conducting channel across the bacterial cytoplasmic membrane. It binds the peripheral ATPase SecA to form the translocase. When isoleucine 278 in transmembrane segment 7 of the SecY subunit was replaced by a unique cysteine, SecYEG supported an increased preprotein translocation and SecA translocation ATPase activity, and allowed translocation of a preprotein with a defective signal sequence. SecY(I278C)EG binds SecA with a higher affinity than normal SecYEG, in particular in the presence of ATP. The increased translocation activity of SecY(I278C)EG was confirmed in a purified system consisting of SecYEG proteoliposomes, while immunoprecipitation in detergent solution reveal that translocase-preprotein complexes are more stable with SecY(I278C) than with normal SecY. These data imply an important role for SecY transmembrane segment 7 in SecA binding. As improved SecA binding to SecY was also observed with the prlA4 suppressor mutation, it may be a general mechanism underlying signal sequence suppression.     

Binding of SecA to the SecYEG complex accelerates the rate of nucleotide exchange on SecA

SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction.     

Demonstration of a specific Escherichia coli SecY-signal peptide interaction

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide-SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA.     

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.     

Binding,activation and dissociation of the dimeric SecA ATPase at the dimeric SecYEG translocase

The bacterial preprotein translocase is comprised of a membrane-embedded oligomeric SecYEG structure and a cytosolic dimeric SecA ATPase. The associations within SecYEG oligomers and SecA dimers, as well as between these two domains are dynamic and reversible. Here, it is shown that a covalently linked SecYEG dimer forms a functional translocase and a high affinity binding site for monomeric and dimeric SecA in solution. The interaction between these two domains stimulates the SecA ATPase, and nucleotides modulate the affinity and ratio of SecA monomers and dimers bound to the linked SecYEG complex. During the translocation reaction, the SecA monomer remains in stable association with a SecYEG protomer and the translocating preprotein. The nucleotides and translocation-dependent changes of SecA-SecYEG associations and the SecA dimeric state may reflect important facets of the preprotein translocation reaction.     

Oligomeric states of the SecA and SecYEG core components of the bacterial Sec translocon

Many proteins synthesized in the cytoplasm ultimately function in non-cytoplasmic locations. In Escherichia coli, the general secretory (Sec) pathway transports the vast majority of these proteins. Two fundamental components of the Sec transport pathway are the SecYEG heterotrimeric complex that forms the channel through the cytoplasmic membrane, and SecA, the ATPase that drives the preprotein to and across the membrane. This review focuses on what is known about the oligomeric states of these core Sec components and how the oligomeric state might change during the course of the translocation of a preprotein.     

Energetics of SecA dimerization

Transport of many proteins to extracytoplasmic locations occurs via the general secretion (Sec) pathway. In Escherichia coli, this pathway is composed of the SecYEG protein-conducting channel and the SecA ATPase. SecA plays a central role in binding the signal peptide region of preproteins, directing preproteins to membrane-bound SecYEG and promoting translocation coupled with ATP hydrolysis. Although it is well established that SecA is crucial for preprotein transport and thus cell viability, its oligomeric state during different stages of transport remains ill defined. We have characterized the energetics of SecA dimerization as a function of salt concentration and temperature and defined the linkage of SecA dimerization and signal peptide binding using analytical ultracentrifugation. The use of a new fluorescence detector permitted an analysis of SecA dimerization down to concentrations as low as 50 nM. The dimer dissociation constants are strongly dependent on salt. Linkage analysis indicates that SecA dimerization is coupled to the release of about five ions, demonstrating that electrostatic interactions play an important role in stabilizing the SecA dimer interface. Binding of signal peptide reduces SecA dimerization affinity, such that K_d increases about 9-fold from 0.28 μM in the absence of peptide to 2.68 μM in the presence of peptide. The weakening of the SecA dimer that accompanies signal peptide binding may poise the SecA dimer to dissociate upon binding to SecYEG.     

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein‐conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster's resonance energy transfer and fluorescence (cross‐) correlation spectroscopy with single‐molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.     

The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity.

SecYEG forms the protein-conducting channel of the Escherichia coli translocase. It binds the peripheral ATPase SecA that drives the preprotein translocation reaction. PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence. The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes. SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4. This activity correlates with an elevated SecA-translocation ATPase and SecA binding affinity. SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity. These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity.     

The oligomeric distribution of SecYEG is altered by SecA and translocation ligands

The multimeric membrane protein complex translocase mediates the transport of preproteins across and integration of membrane proteins into the inner membrane of Escherichia coli. The translocase consists of the peripheral membrane-associated ATPase SecA and the heterotrimeric channel-forming complex consisting of SecY, SecE and SecG. We have investigated the quaternary structure of the SecYEG complex in proteoliposomes. Fluorescence resonance energy transfer demonstrates that SecYEG forms oligomers when embedded in the membrane. Freeze-fracture techniques were used to examine the oligomeric composition under non-translocating and translocating conditions. Our data show that membrane-embedded SecYEG exists in a concentration-dependent equilibrium between monomers, dimers and tetramers, and that dynamic exchange of subunits between oligomers can occur. Remarkably, the formation of dimers and tetramers in the lipid environment is stimulated significantly by membrane insertion of SecA and by the interaction with translocation ligands SecA, preprotein and ATP, suggesting that the active translocation channel consists of multiple SecYEG complexes.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

SecA supports a constant rate of preprotein translocation

In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydrolysis by SecA are thought to drive the stepwise translocation of the preprotein across the membrane. To address how the length of a preprotein substrate affects the SecA-driven translocation process, we constructed derivatives of the precursor of the outer membrane protein A (proOmpA) with 2, 4, 6, and 8 in-tandem repeats of the periplasmic domain. With increasing polypeptide length, an increasing delay in the time before full-length translocation was observed, but the translocation rate expressed as amino acid translocation per minute remained constant. These data indicate that in the ATP-dependent reaction, SecA drives a constant rate of preprotein translocation consistent with a stepping mechanism of translocation.     

Evaluating the oligomeric state of SecYEG in preprotein translocase

SecA insertion and deinsertion through SecYEG drive preprotein translocation at the Escherichia coli inner membrane. We present three assessments of the theory that oligomers of SecYEG might form functional translocation sites. (i) Formaldehyde cross- linking of translocase reveals cross-links between SecY, SecE and SecG, but not higher order oligomers. (ii) Cross-linking of membranes containing unmodified SecE and hemagglutinin-tagged SecE (SecE(HA)) reveals cross-links between SecY and SecE and between SecY and SecE(HA). However, anti-HA immunoprecipitates contain neither untagged SecE nor SecY cross-linked to SecE. (iii) Membranes containing similar amounts of SecE and SecE(HA) were saturated with translocation intermediate (I(29)) and detergent solubilized. Anti-HA immunoprecipitation of I(29) required SecYE(HA)G and SecA, yet untagged SecE was not present in this translocation complex. Likewise, anti-HA immunoprecipitates of membranes containing equal amounts of SecY and SecY(HA) were found to contain SecY(HA) but not SecY. Both immunoprecipitates contain more moles of I(29) than of the untagged subunit, again suggesting that translocation intermediates are not engaged with multiple copies of SecYEG. These studies suggest that the active form of preprotein translocase is monomeric SecYEG.     

Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme.

Escherichia coli preprotein translocase contains a membrane-embedded trimeric complex of SecY, SecE and SecG (SecYEG) and the peripheral SecA protein. SecYE is the conserved functional 'core' of the SecYEG complex. Although sufficient to provide sites for high-affinity binding and membrane insertion of SecA, and for its activation as a preprotein-dependent ATPase, SecYE has only very low capacity to support translocation. The proteins encoded by the secD operon--SecD, SecF and YajC--also form an integral membrane heterotrimeric complex (SecDFyajC). Physical and functional studies show that these two trimeric complexes are associated to form SecYEGDFyajC, the hexameric integral membrane domain of the preprotein translocase 'holoenzyme'. Either SecG or SecDFyajC can support the translocation activity of SecYE by facilitating the ATP-driven cycle of SecA membrane insertion and de-insertion at different stages of the translocation reaction. Our findings show that each of the prokaryote-specific subunits (SecA, SecG and SecDFyajC) function together to promote preprotein movement at the SecYE core of the translocase.     

The PrlA and PrlG phenotypes are caused by a loosened association among the translocase SecYEG subunits.

prlA mutations in the gene encoding the SecY subunit of the membrane domain of the Escherichia coli preprotein translocase confer many phenotypes: enhanced translocation rates, increased affinity for SecA, diminished requirement for functional leader sequences, reduced proton-motive force (PMF) dependence of preprotein translocation and facilitated translocation of preproteins with folded domains. We now report that both prlA and prlG mutations weaken the associations between the SecY, SecE and SecG subunits of the translocase. This loosened association increases the initiation of translocation by facilitating the insertion of SecA with its bound preprotein but reduces the stimulatory effect of the PMF during the initial step of translocation. Furthermore, the originally isolated prlA4 mutant, which possesses a particularly labile SecYEG complex, acquired a secondary mutation that restored the stability while conserving the flexibility of the complex. Combinations of certain prlA and prlG mutations, known to cause synthetic lethality in vivo, dramatically loosen subunit association and lead to complete disassembly of SecYEG. These findings underscore the importance of the loosened SecYEG association for the Prl phenotypes. We propose a model in which each of the PrlA and PrlG phenotypes derive from this enhanced SecYEG conformational flexibility.     

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