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.     

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.     

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.     

A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery

The gene prlA codes for a factor that appears to function in the export of proteins in Escherichia coli. This conclusion is based on the finding that mutations altering the prlA gene product restore export of envelope proteins with defective signal sequences. Previous results showed that the prlA gene lies in an operon (spc) known to code for ten different ribosomal proteins. Our studies show that the prlA gene lies promoter-distal to the last known ribosomal protein gene in this operon. Evidence from gene fusions constructed in vitro suggests that prlA codes for a protein containing at least 300 amino acids. Thus a heretofore unidentified protein specified by a gene within the spc operon appears to be a component of the cellular protein export machinery.     

Streptokinase mutations relieving Escherichia coli K-12 (prlA4) of detriments caused by the wild-type skc gene

A novel phenotype is described for Escherichia coli K-12 carrying the prlA4 allele determining a membrane component of the protein export mechanism. It is manifest as transformation deficiency for plasmids containing the cloned group C streptococcal streptokinase gene, skc. Streptokinase plasmid mutations relieving the prlA4 strain of this deficiency fell into three classes. Class 1 included skc::IS5 insertions, with IS5 integrated in a region encoding the Skc signal sequence and inactivating skc. Class 2 included IS1 insertions leaving skc intact but reducing skc expression, presumably by altering the function of the skc promoter as judged by an insertion site close to the -35 region. The most interesting class, 3, included skc deletions removing the entire signal sequence or a tetrapeptide from its hydrophobic core. The tetrapeptide deletion reduced the size, hydrophobicity, and predicted alpha-helicity of the central region of the Skc signal sequence but facilitated the export of mature Skc in both the wild type and the prlA4 mutant. These findings indicate that the incompatibility between prlA4 and skc is related to deleterious effects of the Skc signal sequence. The tetrapeptide deletion may function by altering the conformation of the signal sequence so as to render interaction with both the PrlA wild-type protein and the PrlA4 mutant protein less detrimental to the export mechanism. These findings also provide an explanation for the difficulties encountered in cloning streptokinase genes in E. coli plasmids and maintaining their structural stability.     

PrlA and PrlG suppressors reduce the requirement for signal sequence recognition.

Selection for suppressors of defects in the signal sequence of secretory proteins has led most commonly to identification of prlA alleles and less often to identification of prlG alleles. These genes, secY/prlA and secE/prlG, encode integral membrane components of the protein translocation system of Escherichia coli. We demonstrate that an outer membrane protein, LamB, that lacks a signal sequence can be exported with reasonable efficiency in both prlA and prlG suppressor strains. Although the signal sequence is not absolutely required for export of LamB, the level of export in the absence of prl suppressor alleles is exceedingly low. Such strains are phenotypically LamB-, and functional LamB can be detected only by using sensitive infectious-center assays. Suppression of the LamB signal sequence deletion is dependent on normal components of the export pathway, indicating that suppression is not occurring through a bypass mechanism. Our results indicate that the majority of the known prlA suppressors function by an identical mechanism and, further, that the prlG suppressors work in a similar fashion. We propose that both PrlA and PrlG suppressors lack a proofreading activity that normally rejects defective precursors from the export pathway.     

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.     

prlA-mediated suppression of signal sequence mutations is modulated by the secA gene product of Escherichia coli K-12.

We studied the dependence of prlA-mediated suppression of signal sequence mutations in maltose-binding protein on cellular SecA levels in Escherichia coli. Reduction of SecA levels within the cell had strong positive and negative effects on prlA-mediated suppression, depending on the particular signal sequence mutations involved. This finding suggests that prlA and secA gene products are both components of a common export system.     

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.     

Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coli.

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564-5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon.     

Novel prlA alleles defective in supporting staphylokinase processing in Escherichia coli

A class of prlA (secY) alleles of Escherichia coli (prlA4-1 and prlA401) which specifically block the export of staphylokinase has been identified (T. Iino and T. Sako, J. Biol. Chem. 263:19077-19082, 1988; T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). To determine more precisely the region in PrlA (SecY) effective for the blockage of processing of the staphylokinase precursor, additional prlA mutants which failed to support processing of the staphylokinase precursor were isolated. Two of the five mutant alleles isolated (secY121 and secY161) complemented the temperature sensitivity of a secY24 strain and had no detectable effect on the processing of endogenous secretory proteins of E. coli. In addition, a staphylokinase mutant having glycine in place of serine at position 17 in its signal sequence relieved the detrimental effect of these mutations. All of these characteristics indicate that these two alleles resemble the prlA4-1 and prlA401 alleles. On the other hand, the remaining three mutant alleles (secY47, secY105, and secY112) had no significant PrlA activity. The mutations of secY121 and secY161 were mapped very close to those of prlA4-1 and prlA401 in the presumed transmembrane segment 7 of PrlA. These results indicate that transmembrane segment 7 of PrlA plays a crucial role in the recognition of the staphylokinase signal sequence.     

Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope.

An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently. Consequently, such a strain is defective for the utilization of maltose as a sole carbon source. We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein. A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E. coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map. Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope. Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur. Our data suggest that proper assembly of a complex is required for efficient export of E. coli envelope proteins to their various extracytoplasmic compartments.     

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation.

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.     

Inhibition and resumption of processing of the staphylokinase in some Escherichia coli prlA suppressor mutants

Escherichia coli strains carrying certain prlA mutations (prlA4 and prlA401) could not support the processing and export of staphylokinase, resulting in the accumulation of the precursor form under high-level synthesis conditions. In order to clarify the cause of the defect in the structure of staphylokinase, we constructed signal peptide mutations of sak which suppressed the processing defect in the prlA4 cells by site-directed mutagenesis. The processing defect was suppressed when glycine or asparagine was introduced in place of the serine residue at position 17 from the amino terminus of the signal peptide. Substitutions of glycine for the leucine residue at position 15 and for the serine residue at position 19 were also effective. Other mutations we constructed had no suppression activity. Taking account of the correlation between the suppression activity and the parameter value of each substituted amino acid for the beta-turn probability, we predict that the staphylokinase signal peptide requires a more bending structure at the end of the hydrophobic core to act efficiently in the prlA4 cells than in the prl+ cells and that a function of the PrlA protein necessary to recognize the staphylokinase signal peptide has become deficient through the prlA4 mutation.     

Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide.

Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process. The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3. It was found that a net positive charge was not absolutely required for MBP export to the periplasm. However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1. The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions. Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events. The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core.     

A Mutation Affecting the Regulation of a Seca-Lacz Fusion Defines a New Sec Gene

It was shown previously that the secA gene of Escherichia coli is derepressed in cells that have a defect in protein export. Here it is demonstrated that the beta-galactosidase produced by a secA-lacZ gene fusion strain is regulated in the same way. Studies on the fusion strain reveal that the promoter or a site involved in regulation of the secA gene is located considerably upstream from the structural gene. The properties of the fusion strain provide a new selection for mutants that are defective in protein export. Selection for increased lac expression of a secA-lacZ fusion strain yields mutations in three of the known sec genes, secA, secD and prlA/secY. In addition, mutations in several genes not previously known to affect secA expression were obtained. A mutation in one of these genes causes a pleiotropic defect in protein export and a cold-sensitive growth defect; this gene, which maps at approximately 90 min on the bacterial chromosome, has been named secE.     

PrlA suppressor mutations cluster in regions corresponding to three distinct topological domains.

The SecY protein of Escherichia coli and its homologues in other organisms, are integral components of the cellular protein translocation machinery. Suppressor mutations that alter SecY (the prlA alleles) broaden the specificity of this machinery and allow secretion of precursor proteins with defective signal sequences. Twenty-five prlA alleles have been characterized. These suppressor mutations were found to cluster in regions corresponding to three distinct topological domains of SecY. Based on the nature and position of the prlA mutations, we propose that transmembrane domain 7 of SecY functions in signal sequence recognition. Results suggest that this interaction may involve a right-handed supercoil of alpha-helices. Suppressor mutations that alter this domain appear to prevent signal sequence recognition, and this novel mechanism of suppression suggests a proofreading function for SecY. We propose that suppressor mutations that alter a second domain of SecY, transmembrane helix 10, also affect this proof-reading function, but indirectly. Based on the synthetic phenotypes exhibited by double mutants, we propose that these mutations strengthen the interaction with another component of the translocation machinery, SecE. Suppressor mutations were also found to cluster in a region corresponding to an amino-terminal periplasmic domain. Possible explanations for this unexpected finding are discussed.     

Thesec andprl genes ofEscherichia coli

Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec,secretion). The other identifies dominant suppressors of signal sequence mutations (prl,proteinlocalization). Subsequent characterization reveals that in at least three cases,prlA/secY,prlD/secA, andprlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.     

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.