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.     

Demonstration in vivo that interaction of maltose-binding protein with SecB is determined by a kinetic partitioning.

An early step in the export of maltose-binding protein to the periplasm is interaction with the molecular chaperone SecB. We demonstrate that binding to SecB in vivo is determined by a kinetic partitioning between the folding of maltose-binding protein to its native state and its association with SecB. A complex of SecB and a species of maltose-binding protein that folds slowly is shown to be longer-lived than a complex of the wild-type maltose-binding protein and SecB. In addition, we show that incomplete nascent chains, which are unable to fold, remain complexed with SecB.     

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.     

The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro.

It has been proposed that the cytoplasmic SecB protein functions as a component of the Escherichia coli protein export machinery by serving as an antifolding factor that retards folding of the precursor maltose-binding protein (preMBP) into a translocation-incompetent form. In this study, it was found that SecB directly interacts with wild-type preMBP and various mutationally altered MBP species synthesized in vitro to form a SecB-MBP complex that can be precipitated with anti-SecB serum. The association of SecB with wild-type preMBP was relatively unstable; such a complex was formed only when SecB was present cotranslationally or after denaturation of previously synthesized preMBP and was detected with only low efficiency. In marked contrast, MBP species that were defective in the ability to assume the stable conformation of wild-type preMBP or that exhibited significantly slower folding kinetics formed much more stable complexes with SecB. In one case, we demonstrated that SecB did not need to be present cotranslationally for complex formation to occur. Formation of a complex between SecB and MBP was clearly not dependent on the MBP signal peptide. However, we were unable to detect complex formation between SecB and MBP lacking virtually the entire signal peptide but having a completely intact mature moiety. This MBP species folded at a rate considerably faster than that of wild-type preMBP. The propensity of this mutant protein to assume the native conformation of mature MBP apparently precludes a stable association with SecB, whereas an MBP species lacking a signal peptide but exhibiting altered folding properties did form a complex with SecB that could be precipitated with anti-SecB serum.     

Interaction of SecB with intermediates along the folding pathway of maltose-binding protein.

SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.     

The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein

Evidence is presented that the E. coli secB gene encodes a soluble protein that interacts with the mature region of the precursor maltose-binding protein (MBP), and promotes MBP export by preventing premature folding of the newly synthesized polypeptide into an export-incompetent form. The interaction of SecB with MBP was indicated by the finding that synthesis of various export-defective MBP species interfered with normal protein export by limiting SecB availability. The antifolding activity of SecB was demonstrated by the following: the defect in MBP export in SecB- cells was suppressed by mutational alterations affecting MBP folding; export of a mutant MBP that is accomplished in a strictly posttranslational mode was totally blocked in SecB- cells; and the rate of folding of wild-type MBP synthesized in vitro was found to be accelerated when SecB was absent and greatly retarded when excess SecB was present.     

SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)⁵ and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.     

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.     

Identification of a Sequence Motif That Confers SecB Dependence on a SecB-Independent Secretory Protein In Vivo

SecB is a cytosolic chaperone which facilitates the transport of a subset of proteins, including membrane proteins such as PhoE and LamB and some periplasmic proteins such as maltose-binding protein, in Escherichia coli. However, not all proteins require SecB for transport, and proteins such as ribose-binding protein are exported efficiently even in SecB-null strains. The characteristics which confer SecB dependence on some proteins but not others have not been defined. To determine the sequence characteristics that are responsible for the SecB requirement, we have inserted a systematic series of short, polymeric sequences into the SecB-independent protein alkaline phosphatase (PhoA). The extent to which these simple sequences convert alkaline phosphatase into a SecB-requiring protein was evaluated in vivo. Using this approach we have examined the roles of the polarity and charge of the sequence, as well as its location within the mature region, in conferring SecB dependence. We find that an insert with as few as 10 residues, of which 3 are basic, confers SecB dependence and that the mutant protein is efficiently exported in the presence of SecB. Remarkably, the basic motifs caused the protein to be translocated in a strict membrane potential-dependent fashion, indicating that the membrane potential is not a barrier to, but rather a requirement for, translocation of the motif. The alkaline phosphatase mutants most sensitive to the loss of SecB are those most sensitive to inhibition of SecA via azide treatment, consistent with the necessity for formation of a preprotein-SecB-SecA complex. Furthermore, the impact of the basic motif depends on location within the mature protein and parallels the accessibility of the location to the secretion apparatus.     

分子伴侣 SecB 基因和人淋巴毒素基因在大肠杆菌中的共表达

分子伴侣ＳｅｃＢ基因和人淋巴毒素基因在大肠杆菌中的共表达周颖张青殷长传宋大新陈永青（复旦大学微生物学系和遗传研究所上海２００４３３）分子伴侣（Ｃｈａｐｅｒｏｎｅ）是细胞内催化及维持其他蛋白质正确构象的一类蛋白质分子［１,２］。研究表明,分子伴...     

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.     

Expression of Escherichia coli SecB in Bacillus subtilis facilitates secretion of the SecB-dependent maltose-binding protein of E. coli.

Less than 20% of the Escherichia coli maltose-binding protein (MBP) synthesized in Bacillus subtilis is exported. However, a portion of the secreted MBP was processed cotranslationally. Coexpression of SecB, a secretion-related chaperone of E. coli, stimulated posttranslational export of MBP in B. subtilis but inhibited its cotranslational processing. Export of a SecB-independent MBP-ribose-binding protein hybrid precursor was not enhanced by SecB. A slowly folding MBP derivative (MBP-Y283D) was more efficiently secreted than wild-type MBP, suggesting that the antifolding activity of SecB promotes posttranslational secretion of MBP in B. subtilis.     

Escherichia coli exports previously folded and biotinated protein domains

Biotination of proteins is a post-translational modification that requires a folded acceptor domain. We previously showed that an acceptor domain fused to the carboxyl terminus of several cytosolic proteins results in biotinated fusion proteins in vivo. We now show that proteins encoded by translational gene fusions of two periplasmic proteins, alkaline phosphatase and TEM beta-lactamase, to carboxyl-terminal biotin-accepting sequences are biotinated and exported by Escherichia coli. Expression of the alkaline phosphatase fusion protein in wild type strains resulted in inefficient biotination of the fusion product. This result was due to the rapid export of the acceptor protein before biotination could occur since a very large increase in biotinated fusion protein levels was observed in strains lacking the SecB chaperone protein. The beta-lactamase fusion protein was biotinated but was only stable in strains lacking the DegP periplasmic protease. Both biotinated fusion proteins accumulated in the culture medium in strains possessing defective outer membranes. These results indicate that the export machinery can accommodate both a post-translational modification and a protein domain previously folded into its mature conformation in vivo.     

Purification of the Escherichia coli secB gene product and demonstration of its activity in an in vitro protein translocation system

Mutations in the Escherichia coli secB gene lead to protein export defects in vivo (Kumamoto, C.A., and Beckwith, J. (1985) J. Bacteriol. 163, 267-274). To demonstrate directly the participation of the secB gene product (SecB) in protein export, SecB was purified, and its effects on in vitro protein translocation were analyzed. SecB was purified from soluble extracts of a strain that overproduced it, by ammonium sulfate precipitation, DEAE-cellulose chromatography, and differential precipitation at acid pH. The chromatographic behavior on gel filtration columns indicated apparent molecular masses of approximately 90 kDa for both purified SecB and SecB in cytosolic extracts of wild type cells. When added to a translocation mixture, purified SecB stimulated pro-OmpA translocation into membrane vesicles. SecB also suppressed the thermoinduced defect in translocating activity of membranes derived from a secY24 mutant. The results of these in vitro studies and of previous in vivo studies demonstrate that SecB plays a direct role in normal protein export in E. coli.     

Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli.

It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export.     

Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli

High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.     

Folded HasA inhibits its own secretion through its ABC exporter

Gram-negative bacterial proteins secreted by ABC exporters carry a secretion signal in their carboxylic extremities. This characteristic suggests that the polypeptide needs to be fully synthesized before it can be secreted and, therefore, presumably may fold at least in part before its secretion. We investigated the relationship between folding and secretion using HasA, a hemoprotein of Serratia marcescens secreted into the extracellular medium by a dedicated Has ABC exporter. We first demonstrated that when HasA is sequestered in the cytoplasm it can acquire its tertiary structure, as assessed from its capacity to bind heme. The cytoplasmic pool of HasA cannot be secreted and inhibits the secretion of newly synthesized molecules. HasA folding in the cytoplasm was independent of either its capacity to bind heme or the presence of SecB, although SecB is essential for HasA secretion. Our findings indicate a strong coupling between synthesis and secretion in the type I secretion pathway.     

In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex.

We characterised the behaviour of the purified precursor protein prePhoE upon dilution from 8 M urea by CD, fluorescence spectroscopy and gel-filtration techniques. It is demonstrated that prePhoE rapidly adopts beta structure, folds and aggregates upon dilution to urea concentrations below 3 M. These processes are paralleled by a loss of translocation competence. Furthermore the interaction of prePhoE with SecB was investigated. SecB is shown to have a very high content of beta structure, therefore we propose that precursor recognition by SecB is mediated through beta-beta interaction. It is shown that SecB has little effect on the adoption of secondary structure and tertiary folding upon dilution of the precursor from urea. However, SecB prevents the precursor from aggregating by forming a functional and stable complex.     

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.