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.     

Covalently dimerized SecA is functional in protein translocation

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.     

Structural determinants of SecB recognition by SecA in bacterial protein translocation

SecB is a bacterial chaperone involved in directing pre-protein to the translocation pathway by its specific interaction with the peripheral membrane ATPase SecA. The SecB-binding site on SecA is located at its C terminus and consists of a stretch of highly conserved residues. The crystal structure of SecB in complex with the C-terminal 27 amino acids of SecA from Haemophilus influenzae shows that the SecA peptide is structured as a CCCH zinc-binding motif. One SecB tetramer is bound by two SecA peptides, and the interface involves primarily salt bridges and hydrogen bonding interactions. The structure explains the importance of the zinc-binding motif and conserved residues at the C terminus of SecA in its high-affinity binding with SecB. It also suggests a model of SecB-SecA interaction and its implication for the mechanism of pre-protein transfer in bacterial protein translocation.     

The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.

The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy-terminus. To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation. It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.     

The structural view of bacterial translocation-specific chaperone SecB: implications for function

SecB is a molecular chaperone that functions in bacterial post-translational protein translocation pathway. It maintains newly synthesized precursor polypeptide chains in a translocation-competent state and guides them to the translocon via its high-affinity binding to the ligand as well as to the membrane-embedded ATPase SecA. Recent advances in elucidating the structures of SecB have enabled the examination of protein function in the structural context. Structures of SecB from both Haemophilus influenzae and Escherichia coli support the early two-subsite polypeptide-binding model. In addition, the detailed molecular interaction between SecB and SecA was revealed by a structure of SecB in complex with the C-terminal zinc-containing domain of SecA. These observations explain the dual role of SecB plays in the translocation pathway, as a molecular chaperone and a specific targeting factor. A model of SecB-SecA complex suggests that the binding of SecA to SecB changes the conformation of the polypeptide binding sites in the chaperone, enabling transfer of precursor polypeptides from SecB to SecA. Recent studies also show the presence of a second zinc-independent SecB binding site in SecA and the new interaction might contribute to the function of SecB.     

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.     

Kinetics and energetics of the translocation of maltose binding protein folding mutants

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.     

Zinc stabilizes the SecB binding site of SecA

The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a metal ion. Treatment of SecA with a zinc chelator resulted in a loss of the stimulatory effect of SecB on the SecA translocation ATPase activity, while the activity could be restored by the addition of ZnCl2. Interaction of SecB with the SecB binding domain of SecA is disrupted by chelators of divalent cations, and could be restored by the addition of Cu2+ or Zn2+. Atomic absorption and electrospray mass spectrometry revealed the presence of one zinc atom per monomeric carboxyl terminus of SecA. It is concluded that the SecB binding domain of SecA is stabilized by a zinc ion that promotes the functional binding of SecB to SecA.     

Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins in Escherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secB missense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.     

Additional in vitro and in vivo evidence for SecA functioning as dimers in the membrane: dissociation into monomers is not essential for protein translocation in Escherichia coli

SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.     

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     

SecB, a molecular chaperone with two faces

SecB is a molecular chaperone unique to the phylum Proteobacteria, which includes the majority of known Gram-negative bacteria of medical, industrial and agricultural significance. SecB is involved in the translocation of secretory proteins across the cytoplasmic membrane. The crystal structure of the Haemophilus influenzae SecB provides new insights into how SecB simultaneously recognizes its two ligands: unfolded preproteins and SecA, the ATPase subunit of the translocase. SecB uses its entire molecular surface for these two functions, but for preprotein release and its own membrane release, SecB relies on the catalytic activity of SecA. This defines SecB as a translocation-specific molecular chaperone.     

Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA

In Escherichia coli , precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo , are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.     

SecB protein stabilizes a translocation-competent state of purified prePhoE protein

Efficient translocation of pure precursor of PhoE protein (prePhoE) could be accomplished in an in vitro system consisting of only inverted Escherichia coli inner membrane vesicles, ATP, and SecA and SecB protein. In this in vitro system SecB and not trigger factor could stabilize a translocation-competent state of prePhoE. In contrast, translocation competency of proOmpA could be induced by both trigger factor and SecB protein, suggesting specificity in interactions between cytosolic factors and precursors in outer membrane protein translocation.     

Correlation between requirement for SecA during export and folding properties of precursor polypeptides

The structural complexity of a ligand in association with the molecular chaperones SecB and SecA was investigated using three species of precursor maltose-binding protein, which differ in their stability as a result of an amino acid substitution in each that affects the rate of folding of the polypeptide. In the presence of high concentrations of both SecB and SecA, the precursors were translocated in vitro with indistinguishable kinetics. However, when SecA was limiting, the translocation was more rapid for precursor species, which had lower stability in the native state relative to the stability of the wild-type precursor. We propose that, when in complex with SecB, precursors can form an element of tertiary structure and that these tertiary contacts are blocked when SecA is bound.     

In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4. 5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and DeltamicroH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.     

SecA functions in vivo as a discrete anti‐parallel dimer to promote protein transport

SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer–dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ‐benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X‐ray structures and assessing their in vivo photo‐crosslinking pattern. A discrete anti‐parallel 1M6N‐like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG‐bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer‐driven translocation mechanism.     

Evidence that SecB enhances the activity of SecA

In Escherichia coli, SecA is a critical component of the protein transport machinery which powers the translocation process by hydrolyzing ATP and recognizing signal peptides which are the earmark of secretory proteins. In contrast, SecB is utilized by only a subset of preproteins to prevent their premature folding and chaperone them to membrane-bound SecA. Using purified components and synthetic signal peptides, we have studied the interaction of SecB with SecA and with SecA-signal peptide complexes in vitro. Using a chemical cross-linking approach, we find that the formation of SecA-SecB complexes is accompanied by a decrease in the level of cross-linking of SecA dimers, suggesting that SecB induces a conformational change in SecA. Furthermore, functional signal peptides, but not dysfunctional ones, promote the formation of SecA-SecB complexes. SecB is also shown to directly enhance the ATPase activity of SecA in a concentration-dependent and saturable manner. To determine the biological consequence of this finding, the influence of SecB on the signal peptide-stimulated SecA/lipid ATPase was studied using synthetic peptides of varying hydrophobicity. Interestingly, the presence of SecB can sufficiently boost the response of signal peptides with moderate hydrophobicity such that it is comparable to the activity generated by a more hydrophobic peptide in the absence of SecB. The results suggest that SecB directly enhances the activity of SecA and provide a biochemical basis for the enhanced transport efficiency of preproteins in the presence of SecB in vivo.     

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.     

Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.