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.     

Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.     

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.     

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.     

Sec-translocase mediated membrane protein biogenesis

Alpha-helical transmembrane proteins in bacteria are localized within the plasma membrane. The membrane assembly of these proteins requires protein devices for insertion into the lipid bilayer. In E. coli, membrane proteins require the SRP pathway components Ffh, 4.5S RNA and FtsY for membrane targeting and the SecYEGDF translocase and, in some cases, SecA, for translocation of hydrophilic domains. In addition, YidC, a recently discovered membrane protein, mediates the membrane integration and folding of hydrophobic domains of membrane proteins. In this review, we will describe the current status of the protein targeting and membrane integration pathways.     

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.     

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.     

Export of beta-lactamase is independent of the signal recognition particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.     

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.     

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.     

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.     

Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.     

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.     

Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.     

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.     

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.     

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.     

Orientation of SecA and SecB in complex, derived from disulfide cross-linking

SecA is the ATPase that acts as the motor for protein export in the general secretory, or Sec, system of Escherichia coli. The tetrameric cytoplasmic chaperone SecB binds to precursors of exported proteins before they can become stably folded and delivers them to SecA. During this delivery step, SecB binds to SecA. The complex between SecA and SecB that is maximally active in translocation contains two protomers of SecA bound to a tetramer of SecB. The aminoacyl residues on each protein that are involved in binding the other have previously been identified by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy; however, that study provided no information concerning the relative orientation of the proteins within the complex. Here we used our extensive collection of single-cysteine variants of the two proteins and subjected pairwise combinations of SecA and SecB to brief oxidation to identify residues in close proximity. These data were used to generate a model for the orientation of the two proteins within the complex.     

SecA protein is directly involved in protein secretion in Escherichia coli

A high-expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.     

The structural basis of protein targeting and translocation in bacteria

In Gram-negative bacteria, two distinct targeting routes assist in the proper localization of secreted and membrane proteins. Signal recognition particle (SRP) mainly targets ribosome-bound nascent membrane proteins, whereas SecB facilitates the targeting of periplasmic and outer membrane proteins. These routes converge at the translocase, a protein-conducting pore in the membrane that consists of the SecYEG complex associated with the peripheral ATPase, SecA. Recent structural studies of the targeting and the translocating components provide insights into how substrates are recognized and suggest a mechanism by which proteins are transported through an aqueous pore in the cytoplasmic membrane.