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.     

Regions of maltose-binding protein that influence SecB-dependent and SecA-dependent export in Escherichia coli.

In Escherichia coli, the efficient export of maltose-binding protein (MBP) is dependent on the chaperone SecB, whereas export of ribose-binding protein (RBP) is SecB independent. To localize the regions of MBP involved in interaction with SecB, hybrids between MBP and RBP in SecB mutant cells were constructed and analyzed. One hybrid consisted of the signal peptide and first third of the mature moiety of MBP, followed by the C-terminal two-thirds of RBP (MBP-RBP112). This hybrid was dependent upon SecB for its efficient export and exhibited a strong export defect in secA mutant cells. A hybrid between RBP and MBP with the same fusion point was also constructed (RBP-MBP116). The RBP-MBP116 hybrid remained SecB independent and only exhibited a partial export defect in secA mutant cells. In addition, MBP species with specific alterations in the early mature region were less dependent on SecB for their efficient export. The export of these altered MBP species was also less affected in secA mutant cells and in cells treated with sodium azide. These results present additional evidence for the targeting role of SecB.     

SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins.

The efficient export of the Escherichia coli maltose-binding protein (MBP) is known to be SecB dependent, whereas ribose-binding protein (RBP) export is SecB independent. When the MBP and RBP signal peptides were exchanged precisely at the signal peptidase processing sites, the resultant RBP-MBP and MBP-RBP hybrid proteins both were efficiently exported in SecB+ cells. However, only MBP-RBP was efficiently exported in SecB- cells; RBP-MBP exhibited a significant export defect, a finding that was consistent with previous proposals that SecB specifically interacts with the mature moiety of precursor MBP to promote export. The relatively slow, totally posttranslational export mode exhibited by certain mutant RBP and MBP-RBP species in SecB+ cells was not affected by the loss of SecB. In contrast, MBP and RBP-MBP species with similarly altered signal peptides were totally export defective in SecB- cells. Both export-defective MBP and RBP-MBP interfered with SecB-mediated protein export by depleting cells of functional SecB. In contrast, neither export-defective RBP nor MBP-RBP elicited such an interference effect. These and other data indicated that SecB is unable to interact with precursor RBP or that any interaction between these two proteins is considerably weaker than that of SecB with precursor MBP. In addition, no correlation could be established between a SecB requirement for export and PrlA-mediated suppression of signal peptide export defects. Finally, previous studies have established that wild-type MBP export can be accomplished cotranslationally, whereas wild-type RBP export is strictly a posttranslational process. In this study, cotranslational export was not detected for either MBP-RBP or RBP-MBP. This indicates that the export mode exhibited by a given precursor protein (cotranslational versus posttranslational) is determined by properties of both the signal peptide and the mature moiety.     

A mutation of Escherichia coli SecA protein that partially compensates for the absence of SecB.

The Escherichia coli SecB protein is a cytosolic chaperone protein that is required for rapid export of a subset of exported proteins. To aid in elucidation of the activities of SecB that contribute to rapid export kinetics, mutations that partially suppressed the export defect caused by the absence of SecB were selected. One of these mutations improves protein export in the absence of SecB and is the result of a duplication of SecA coding sequences, leading to the synthesis of a large, in-frame fusion protein. Unexpectedly, this mutation conferred a second phenotype. The secA mutation exacerbated the defective protein export caused by point mutations in the signal sequence of pre-maltose-binding protein. One explanation for these results is that the mutant SecA protein has sustained a duplication of its binding site(s) for exported protein precursors so that the mutant SecA is altered in its interaction with precursor molecules.     

Directed evolution of efficient secretion in the SRP-dependent export of TolB

Signal sequence non-optimal codons have been shown to be important for the folding and efficient export of maltose binding protein (MBP), a SecB dependent protein. In this study, we analysed the importance of signal sequence non-optimal codons of TolB, a signal recognition particle (SRP) dependent exported protein. The protein production levels of wild type TolB (TolB-wt) and a mutant allele of TolB in which all signal sequence non-optimal codons were changed to a synonymous optimal codon (TolB-opt), revealed that TolB-opt production was 12-fold lower than TolB-wt. This difference could not be explained by changes in mRNA levels, or plasmid copy number, which was the same in both strains. A directed evolution genetic screen was used to select for mutants in the TolB-opt signal sequence that resulted in higher levels of TolB production. Analysis of the 46 independent TolB mutants that reverted to wild type levels of expression revealed that at least four signal sequence non-optimal codons were required. These results suggest that non-optimal codons may be required for the folding and efficient export of all proteins exported via the Sec system, regardless of whether they are dependent on SecB or SRP for delivery to the inner membrane.     

The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export.

The product of the secB gene is required for export of a subset of secreted proteins to the outer membrane and periplasm of Escherichia coli. Precursor maltose-binding protein (MBP) accumulates in the cytoplasm of secB-carrying mutants, but export of alkaline phosphatase is only minimally affected by secB mutations. When export of MBP-alkaline phosphatase hybrid proteins was analyzed in wild-type and secB-carrying mutant strains, the first third of mature MBP was sufficient to render export of the hybrid proteins dependent on SecB. Substitution of a signal sequence from a SecB-independent protein had no effect on SecB-dependent export. These findings show that the first third of mature MBP is capable of conferring export incompetence on an otherwise competent protein.     

Biogenesis of outer membrane protein PhoE of Escherichia coli. Evidence for multiple SecB-binding sites in the mature portion of the PhoE protein.

Efficient in vivo translocation of the precursor of Escherichia coli outer membrane protein PhoE across the inner membrane is shown to depend on SecB protein. A set of mutants, carrying internal deletions in the phoE gene, was used to locate a possible SecB-binding site and/or a site that makes the protein dependent on SecB for export. Except for two small mutant PhoE proteins, the in vivo and in vitro translocation of all mutant proteins was more efficient in the presence of SecB. The interaction of SecB protein with wild-type and mutant PhoE proteins, synthesized in vitro, was further studied in co-immunoprecipitation experiments with anti-SecB protein serum. The efficiencies of co-immunoprecipitation of precursor and mature PhoE were very similar, indicating the absence of a SecB-binding site in the signal sequence. Moreover, all mutant proteins with deletions in the mature moiety of the PhoE protein were co-immunoprecipitated in these assays, albeit mostly with reduced efficiency. Taken together, these results indicate the existence of multiple SecB-binding sites in the mature portion of the PhoE protein.     

PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane

The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.     

A new suppressor of a lamB signal sequence mutation, prlZ1, maps to 69 minutes on the Escherichia coli chromosome.

Reversion analysis has been employed to isolate suppressors that restore export of a unique LamB signal sequence mutant. The mutation results in a substitution of Arg for Met at position 19, which prevents LamB export to the outer membrane and leads to a Dex- phenotype. Unlike other LamB signal sequence mutants utilized for reversion analysis, LamB19R becomes stably associated with the inner membrane in an export-specific manner. In this study, Dex+ revertants were selected and various suppressors were isolated. One of the extragenic suppressors, designated prlZ1, was chosen for further study. prlZ1 maps to 69 min on the Escherichia coli chromosome. The suppressor is dominant and SecB dependent. In addition to its effect on lamB19R, prlZ1 suppresses the export defect of signal sequence point mutations at positions 12, 15, and 16, as well as several point mutations in the maltose-binding protein signal sequence. prlZ1 does not suppress deletion mutations in either signal sequence. This pattern of suppression can be explained by interaction of a helical LamB signal sequence with the suppressor.     

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.     

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.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.     

SecB protein: A cytosolic export factor that associates with nascent exported proteins

Soluble factors participate in protein translocation across a variety of biological membranes. TheEscherichia coli soluble protein SecB (the product of thesecB gene) is involved in the export of periplasmic and outer membrane proteins. The isolation ofsecB mutations permitted the demonstration that SecB is required for rapid and efficient export of certain proteins. Consistent with the results of these genetic studies, purified SecB has been shown to stimulate protein translocation acrossE. coli inner membrane vesiclesin vitro. This article presents a review of these past studies of SecB, speculation on the role of SecB in protein translocation, and a comparison of SecB and other factors, trigger factor and GroEL.     

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.     

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.     

Post-translational export of maltose-binding protein in Escherichia coli strains harboring malE signal sequence mutations and either prl+ or prl suppressor alleles

We have studied the export kinetics of the maltose-binding protein (MBP) of Escherichia coli, the malE gene product, when it is synthesized with either a wildtype signal sequence or with a mutationally altered signal sequence that affects the efficiency of secretion to the periplasm. Our results confirm a very rapid export process for the wild-type protein and, in contrast, reveal a relatively slow post-translational mode of export for the altered precursor species. For each different signal sequence mutant, a fraction of the precursor MBP pool that is proportional to the strength of the export defect appears to never exit the cytoplasm. We have also analyzed MBP export in strains harboring prl mutations that suppress malE signal sequence mutations and are thought to somehow alter the specificity of the cell's protein export machinery. The introduction of different prl alleles has no apparent effect on wild-type MBP export but increases both the amount of mutant MBP that is exported and the rate at which this is accomplished. In fact, the presence of two different prl alleles in the same strain can act synergistically in suppressing MBP export defects. The inhibition of total protein synthesis with chloramphenicol can also increase the proportion of pMBP that is post-translationally exported in these strains. A model that describes the initial steps in MBP export is presented.     

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

The signal sequence of the murine serine protease inhibitor PAI-2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild-type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI-2 signal sequence. Using a malE signal sequence mutant, which has a Mal-phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele-specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG.     

Peculiar properties of DsbA in its export across the Escherichia coli cytoplasmic membrane

Export of DsbA, a protein disulfide bond-introducing enzyme, across the Escherichia coli cytoplasmic membrane was studied with special reference to the effects of various mutations affecting translocation factors. It was noted that both the internalized precursor retaining the signal peptide and the periplasmic mature product fold rapidly into a protease-resistant structure and they exhibited anomalies in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in that the former migrated faster than the latter. The precursor, once accumulated, was not exported posttranslationally. DsbA export depended on the SecY translocon, the SecA ATPase, and Ffh (signal recognition particle), but not on SecB. SecY mutations, such as secY39 and secY205, that severely impair translocation of a number of secretory substrates by interfering with SecA actions only insignificantly impaired the DsbA export. In contrast, secY125, affecting a periplasmic domain and impairing a late step of translocation, exerted strong export inhibition of both classes of proteins. These results suggest that DsbA uses not only the signal recognition particle targeting pathway but also a special route of translocation through the translocon, which is hence suggested to actively discriminate pre-proteins.     

prlA suppression of defective export of maltose-binding protein in secB mutants of Escherichia coli.

An Escherichia coli strain containing a signal sequence mutation in the periplasmic maltose-binding protein (MBP) (malE18-1) and a point mutation in the soluble export factor SecB (secBL75Q) is completely defective in export of MBP and unable to grow on maltose (Mal- phenotype). We isolated 95 spontaneous Mal+ revertants and characterized them genetically. Three types of extragenic suppressors were identified: informational (missense) suppressors, a bypass suppressor conferring the Mal+ phenotype in the absence of MBP, and suppressors affecting the prlA gene, which encodes a component of the protein export apparatus. In this study, a novel prlA allele, designated prlA1001 and mapping in the putative second transmembrane domain of the PrlA (SecY) protein, was found. In addition, we isolated a mutation designated prlA1024 which is identical to prlA4-2, the mutation responsible for the signal sequence suppression in the prlA4 (prlA4-1 prlA4-2) double mutant (T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). Comparison of the prlA1024 mutant and the prlA4 double mutant provides a possible explanation for the isolation of these prlA alleles.