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.     

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.     

Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.     

Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.     

Protein localization in E. coli: Is there a common step in the secretion of periplasmic and outer-membrane proteins?

An E. coli strain carrying a fusion of the malE and lacZ genes is induced for the synthesis of a hybrid protein, consisting of the N-terminal part of the maltose-binding protein and the enzymatically active C-terminal part of β-galactosidase, by addition of maltose to cells. The secretion of the protein is initiated by the signal peptide attached to the N terminus of the maltose-binding protein sequence, but is not completed, presumably because the β-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the cytoplasmic membrane. Thus the protein becomes stuck to the cytoplasmic membrane. Under such conditions, periplasmic proteins, including maltose-binding protein (encoded by the malE gene) and alkaline phosphatase, and the major outer-membrane proteins, including OmpF, OmpA and probably lipoprotein, are synthesized as precursor forms with unprocessed signal sequences. This effect is observed within 15 min after high levels of induction are achieved. The simplest explanation for these results and those of pulse-chase experiments is that specific sites in the cytoplasmic membrane become progressively occupied by the hybrid protein, resulting in an inhibition of normal localization and processing of periplasmic and outer-membrane proteins. These results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme. If this interpretation is correct, we can estimate that an E. coli cell has roughly 2 × 10⁴ such sites in the cytoplasmic membrane. A system is described for detecting the precursor of any exported protein.     

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.     

Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins

We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.     

Diverse effects of the MalE-LacZ hybrid protein on Escherichia coli cell physiology.

The hybrid protein between the periplasmic maltose-binding protein and the cytoplasmic beta-galactosidase (the MalE-LacZ hybrid protein) was previously shown to block the export of envelope proteins when synthesized in large amounts. Now we show that the hybrid protein exerts another major effect on the cell, that is, induction of the heat shock proteins. This latter effect was dependent on the htpR gene product but independent of the function of the signal sequence on the hybrid protein. On the other hand, the previously reported induction of the SecA protein by the hybrid protein was independent of htpR and may be caused by the reduced protein export ability of the cell. The functional htpR gene is essential for viability of the cell in which the basal level of the hybrid protein is synthesized, whereas in the absence of the hybrid protein htpR is dispensable at low temperature. These results indicate that the hybrid protein somehow generates a signal or stress that is similar to what the cell experiences at elevated temperatures.     

Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli

A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E. coli cell. The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P. Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

Export and processing of MalE-LacZ hybrid proteins in Escherichia coli

Five classes of MalE-LacZ hybrid proteins have previously been characterized. These proteins differ in the amount of the maltose-binding protein (MBP) that is attached to beta-galactosidase. Although none of these proteins is secreted into the periplasm, the four larger classes of hybrid proteins, those that include an intact MBP signal peptide, are inserted into the cytoplasmic membrane, suggesting that the secretion process has at least been initiated. In this study, we demonstrated that some portion of the four larger hybrid proteins can be translocated across the cytoplasmic membrane, thus permitting processing of the signal peptide. We have found that hybrid proteins that include only a small portion of the mature MBP are inefficiently recognized as exported proteins, and translocation and processing of these appear to be relatively slow, posttranslational events. In marked contrast, hybrid proteins that include a substantial portion of the mature MBP are efficiently recognized, and translocation and processing of these occur very rapidly, possibly cotranslationally. Our results complement other studies and very strongly suggest a role for the mature MBP in the export process.     

Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation

Accessibility of nascent chains of periplasmic proteins to externally added proteinase K was used as the criterion for translocation of polypeptides across the cytoplasmic membrane of E. coli during the process of export. It is concluded for maltose-binding protein and ribose-binding protein that nascent chains carrying the signal sequence are not accessible to the proteinase while chains that have been matured span the membrane and are degraded. Translocation of polypeptides is a late event relative to extent of elongation, occurring only after maltosebinding protein has reached molecular weight 33,000 (80% of its entire length) and after ribosebinding protein has been fully elongated (molecular weight 29,000). The data presented here are inconsistent with postulated mechanisms of export requiring a strict coupling of translocation to elongation of nascent polypeptide chains. In contrast, the data support the idea that entire domains of polypeptides are transferred after their synthesis. This is the case whether the translocation of a protein is initiated post-translationally or begins before synthesis of the entire protein is completed.     

Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space

Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal-sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.     

Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end.

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.     

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.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Experimental confirmation of a key role for non-optimal codons in protein export

Non-optimal codons are defined by low usage and low abundance of corresponding tRNA, and have an established role in translational pausing to allow the correct folding of proteins. Our previous work reported a striking abundance of non-optimal codons in the signal sequences of secretory proteins exported via the sec-dependent pathway in Escherichia coli. In the current study the signal sequence of maltose-binding protein (MBP) was altered so that non-optimal codons were substituted with the most optimal codon from their synonymous codon family. The expression of MBP from the optimized allele (malE-opt) was significantly less than wild-type malE. Expression of MBP from malE-opt was partially restored in a range of cytoplasmic and periplasmic protease deficient strains, confirming that reduced expression of MBP in malE-opt was due to its preferential degradation by cytoplasmic and periplasmic proteases. These data confirm a novel role for non-optimal codon usage in secretion by slowing the rate of translation across the N-terminal signal sequence to facilitate proper folding of the secreted protein.     

Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli.

A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.     

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca , the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE–PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE–PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE–PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fatty-acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli , indicating the possible existence of a family of PulS-like chaperone proteins.