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.     

Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor

Besides SecA and SecB, Escherichia coli cells possess a signal recognition particle (SRP) to target exported proteins to the SecY translocon. Using chemical and site-specific cross-linking in vitro, we show that SRP recognizes the first signal anchor sequence of a polytopic membrane protein (MtlA) resulting in cotranslational targeting of MtlA to SecY and phospholipids of the plasma membrane. In contrast, a possible interaction of SRP with the secretory protein pOmpA is prevented by the association of trigger factor with nascent pOmpA. Trigger factor also prevents SecA from binding to the first 125 amino acids of pOmpA when they are still associated with the ribosome. Under no experimental conditions was SecA found to interact with MtlA. Likewise, virtually no binding of trigger factor to ribosome-bound MtlA occurs even in the complete absence of SRP. Collectively, our results indicate that at the stage of nascent polypeptides, polytopic membrane proteins are selected by SRP for co-translational membrane targeting, whereas secretory proteins are directed into the SecA/SecB-mediated post-translational targeting pathway by means of their preferential recognition by trigger factor.     

Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.     

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.     

SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein.

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.     

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.

The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.     

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.     

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 functions as a cytosolic signal recognition factor for protein export in E. coli

A purified 64 kd protein, consisting of four identical subunits of the 16 kd SecB, binds to the signal sequence of preproteins prior to their translocation across inverted vesicles (INV) derived from the E. coli plasma membrane. The purified SecB tetramer competes with canine signal recognition particle (SRP) in signal sequence binding and thus behaves as a prokaryotic equivalent of SRP. As shown by cell fractionation and immunoblot analysis with anti-SecB antibodies, SecB is a cytosolic protein. An E. coli supernatant depleted of SecB after passage through an anti-SecB Sepharose column retains full translation activity but is unable to support translocation into added INV. Translocation into INV is fully restored by readdition of purified 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.     

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.     

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.     

DnaK promotes the selective export of outer membrane protein precursors in SecA-deficient Escherichia coli

Consistent with many other results indicating that SecA plays an essential role in the translocation of presecretory proteins across the Escherichia coli inner membrane, we previously found that a approximately 95% depletion of SecA completely blocks the export of periplasmic proteins in vivo. Surprisingly, we found that about 25% of the outer membrane protein (OMP) OmpA synthesized after SecA depletion was gradually translocated across the inner membrane. In this study we analyzed the export of several other OMPs after SecA depletion. We found that 25-50% of each OMP as well as an OmpA-alkaline phosphatase fusion protein was exported from SecA-deficient cells. This partial export was completely abolished by the SecA inhibitor sodium azide and therefore still required the participation of SecA. Examination of a variety of OmpA derivatives, however, ruled out the possibility that OMPs are selectively translocated in SecA-deficient cells because SecA binds to their N termini with unusually high affinity. Export after SecA depletion was observed in cells that lack SecB, the primary targeting factor for OMPs, but was abolished by partial inactivation of DnaK. Furthermore, OmpA could be isolated in a stable complex with DnaK. The data strongly suggest that OMPs require only a relatively low level of translocase activity to cross the inner membrane because they can be preserved in a prolonged export-competent state by DnaK.     

Determinants of membrane-targeting and transmembrane translocation during bacterial protein export.

We have separately analyzed membrane-targeting and membrane translocation of an exported bacterial protein. The precursor of the outer membrane protein LamB of Escherichia coli was synthesized in vitro and translocated into inverted plasma membrane vesicles under co- and post-translational conditions. The translation/translocation products of LamB were subsequently resolved into soluble and membrane-associated material. Dissipation of the H(+)-motive force, depletion of ATP and treatment of membranes with N-ethylmaleimide each inhibited processing and translocation of preLamB without preventing its binding to the membranes. Hence, all three conditions block transmembrane passage rather than membrane-targeting. The latter was abolished by pretreatment of salt-extracted membrane vesicles with trypsin. It was also drastically reduced when preLamB was synthesized in cell extracts derived from either a secA amber or a secB null mutant. Membrane-targeting of preLamB therefore requires soluble SecA and SecB as well as a protease-sensitive membrane receptor. The finding that SecA is involved in targeting whereas ATP is required for the transmembrane passage suggests that SecA, which harbors an ATPase activity [Lill et al. (1989), EMBO J., 8, 961-966], might have a dual function in bacterial protein export.     

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.