Requirement of the SecB chaperone for export of a non-secretory polypeptide in Escherichia coli

Summary The SecB protein of Escherichia coli is a cytosolic component of the export machinery which can prevent some precursors from prematurely folding into export-incompatible conformations by binding to the newly synthesised polypeptide. The feature(s) of target proteins recognised by SecB, however, are unclear and have been a matter of controversy. Also, it has not been asked if binding of SecB is specific for secretory proteins. We demonstrate here that a non-secretory polypeptide, a fragment of a tail fiber protein of phage T4, fused to the signal peptide of the outer membrane protein OmpA has a very strong SecB requirement for export and that the signal peptide itself cannot, at least not alone, be responsible for this action of SecB. The data reported, together with those of the literature, suggest that SecB recognizes the polypeptide backbone of the target protein.     

A highly mobile C-terminal tail of the Escherichia coli protein export chaperone SecB.

The Escherichia coli export chaperone SecB binds nascent precursors of certain periplasmic and outer membrane proteins and prevents them from folding or aggregating in the cytoplasm. In this study, we demonstrate that the C-terminal 13 residues of SecB were highly mobile using (1)H NMR spectroscopy. A protein lacking the C-terminal 13 amino acids of wild-type SecB was found to retain the ability to bind unfolded maltose-binding protein (MBP) in vitro but to interfere with the normal kinetics of pre-MBP export when overexpressed in vivo. The defect in export was reversed by overproduction of the peripheral membrane ATPase SecA. Therefore, deletion of the mobile region of SecB may alter the interactions of SecB with SecA.     

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.     

Defining the role of the Escherichia coli chaperone SecB using comparative proteomics

To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by "classical" pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase.     

Export of periplasmic galactose-binding protein in Escherichia coli depends on the chaperone SecB.

The efficient export of galactose-binding protein to the periplasm of Escherichia coli is shown to be dependent on the presence of the cytosolic chaperone SecB.     

Involvement of SecB, a chaperone, in the export of ribose-binding protein.

Ribose-binding protein (RBP) is an exported protein of Escherichia coli that functions in the periplasm. The export of RBP involves the secretion machinery of the cell, consisting of a cytoplasmic protein, SecA, and the integral membrane translocation complex, including SecE and SecY. SecB protein, a chaperone known to mediate the export of some periplasmic and outer membrane proteins, was previously reported not to be involved in RBP translocation even though small amounts of in vitro complexes between SecB and RBP have been detected. In our investigation, it was shown that a dependence on SecB could be demonstrated under conditions in which export was compromised. Species of RBP which carry two mutations, one in the leader that blocks export and a second in the mature protein which partially suppresses the export defect, were shown to be affected by SecB for efficient translocation. Five different changes which suppress the effect of the signal sequence mutation -17LP are all located in the N domain of the tertiary structure of RBP. All species of RBP show similar interaction with SecB. Furthermore, a leaky mutation, -14AE, generated by site-specific mutagenesis causes reduced export in the absence of SecB. These results indicate that SecB can interact with RBP during secretion, although it is not absolutely required under normal circumstances.     

Effect of SecB chaperone on production of periplasmic penicillin acylase in Escherichia coli.

The effect of SecB chaperone on production of periplasmic penicillin acylase (PAC) in Escherichia coli was investigated. It appears that formation of PAC required the function of SecB chaperone and the amount of SecB required was at a basal level. The secB mutant was defective in production of PAC, and the impairment could be complemented by extrachromosomally supplementing SecB in trans. The function of SecB might be primarily stabilizing the cytoplasmic PAC precursors. Overproduction of SecB chaperone usually resulted in an increase in the amount of PAC precursors without enhancing PAC activity. In addition, most of the PAC precursors were located in the periplasm, suggesting that formation of active PAC was likely limited by periplasmic processing steps.     

Influence of impaired chaperone or secretion function on SecB production in Escherichia coli.

The efficient export of proteins through the cytoplasmic membrane of Escherichia coli requires chaperones to maintain protein precursors in a translocation-competent conformation. In addition to SecB, the major chaperone facilitating export of particular precursors, heat shock-induced chaperones DnaK-DnaJ and GroEL-GroES are also involved in this process. By use of secB'-lacZ gene fusions and immunoprecipitation experiments, SecB production was studied in E. coli strains containing conditional lethal mutations in chaperone or sec genes. While the loss of heat shock chaperones resulted in an increased production of SecB, mutations in sec genes showed only minor effects on SecB synthesis. Neither the plasmid-mediated overexpression of precursors of exoproteins nor the overexpression of secB altered the synthesis of SecB. These results suggest that under conditions where chaperones become depleted, E. coli responds by raising the expression of secB. These data confirm the supposed synergy of different chaperones involved in protein export.     

Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants.

Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway. An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export. The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence. Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined. Most of the mutations caused severe defects in RBP export. In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB. Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed. Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export. However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB. These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.     

Export chaperone SecB uses one surface of interaction for diverse unfolded polypeptide ligands

SecB, a remarkable chaperone involved in protein export, binds diverse ligands rapidly with high affinity and low specificity. Site‐directed spin labeling and electron paramagnetic resonance spectroscopy were used to investigate the surface of interaction on the export chaperone SecB. We examined SecB in complex with the unfolded precursor form of outer membrane protein OmpA as well as with a truncated version of OmpA that includes the transmembrane domain and lacks both the signal peptide and the periplasmic domain. In addition, we studied the binding of SecB to the unfolded mature form of galactose‐binding protein, a soluble periplasmic protein. We have previously used the same strategy to map the binding surface for the precursor of galactose‐binding protein. We show that for all ligands tested the patterns of contact are the same.     

Carbon source-dependent synthesis of SecB, a cytosolic chaperone involved in protein translocation across Escherichia coli membranes.

SecB is a cytosolic chaperone involved in protein translocation across cytoplasmic membranes in Escherichia coli. It has been shown to be required for efficient translocation of a subset of precursor proteins but is not essential for cell viability. This study investigated whether synthesis of SecB is growth rate dependent. Interestingly, the total amount of SecB synthesized in the cells was relatively small. Moreover, the levels of SecB were found to be carbon source dependent since more SecB was produced in cells grown in glycerol media than in cells grown in glucose media, regardless of the growth rate. This is in contrast to the other Sec proteins, whose synthesis is growth rate dependent and not related to glucose as a carbon source. In addition, cyclic AMP (cAMP) partially relieves the lower levels of SecB observed in glucose medium, a compensatory effect that depends on the presence of both cya and crp gene products. Thus, the glucose-dependent synthesis of SecB may be related to the cAMP-cAMP receptor protein complex-mediated activation.     

Crystal structure of SecB from Escherichia coli

The chaperone SecB from Escherichia coli is primarily involved in passing precursor proteins into the Sec system via specific interactions with SecA. The crystal structure of SecB from E. coli has been solved to 2.35 A resolution. The structure shows flexibility in the crossover loop and the helix-connecting loop, regions that have been implicated to be part of the SecB substrate-binding site. Moreover conformational variability of Trp36 is observed as well as different loop conformations for the different monomers. Based on this, we speculate that SecB can regulate the access or extent of its hydrophobic substrate-binding site, by modulating the conformation of the crossover loop and the helix-connecting loop. The structure also clearly explains why the tetrameric equilibrium is shifted towards the dimeric state in the mutant SecBCys76Tyr. The buried cysteine residue is crucial for tight packing, and mutations are likely to disrupt the tetramer formation but not the dimer formation.     

Effect of export-specific cytoplasmic chaperone, protein SecB, on secretion of Escherichia coli alkaline phosphatase

The efficiency of secretion of Escherichia coli alkaline phosphatase depends on the presence in cells of a cytoplasmic chaperone—protein SecB. Secretion increases in the presence of this chaperone at 30°C, which is the most favorable for the interaction of SecB with the export-initiation domain found previously in the N-terminal region of the mature enzyme. This interaction most likely occurs in the region of the export domain, which is located close to the signal peptide and in complex with a translocational ATPase—protein SecA.     

Sites of interaction between SecA and the chaperone SecB, two proteins involved in export

SecB, a small tetrameric cytosolic chaperone in Escherichia coli, facilitates the export of precursor poly-peptides by maintaining them in a nonnative conformation and passing them to SecA, which is a peripheral member of the membrane-bound translocation apparatus. It has been proposed by several laboratories that as SecA interacts with various components along the export pathway, it undergoes conformational changes that are crucial to its function. Here we report details of molecular interactions between SecA and SecB, which may serve as conformational switches. One site of interaction involves the final C-terminal 21 amino acids of SecA, which are positively charged and contain zinc. The C terminus of each subunit of the SecA dimer makes contact with the flat beta-sheet that is formed by each dimer of the SecB tetramer. Here we demonstrate that a second interaction exists between the extreme C-terminal alpha-helix of SecB and a site on SecA, as yet undefined but different from the C terminus of SecA. We investigated the energetics of the interactions by titration calorimetry and characterized the hydrodynamic properties of complexes stabilized by both interactions or each interaction singly using sedimentation velocity centrifugation.     

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.     

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Periplasmic secretion has been used in attempts to construct an efficient whole‐cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin‐arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole‐cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole‐cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL‐GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole‐cell OPH activity of the chaperone–coexpressing cells was ～5.5‐fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat‐based periplasmic translocation of OPH in the chaperone–coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole‐cell activity of periplasmic OPH‐secreting cells, yielding an effective whole‐cell biocatalyst system with highly reduced diffusion limitations. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 925–930, 2012     

Substrate specificity of the SecB chaperone

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is approximately 9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20-30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.     

Crystallization of the chaperone protein SecB.

The secretory protein SecB found in Escherichia coli is a molecular chaperone that binds to precursor forms of a number of proteins targeted for export to the periplasmic space. SecB maintains these proteins in a translocation-competent conformation facilitating the translocation process. The material has been cloned and expressed in E. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensions a = 56.0 A, b = 111.1 A, c = 134.7 A, and beta = 104 degrees. The crystals diffract to 8 A resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.