Characterization of a region in mature LamB protein that interacts with a component of the export machinery of Escherichia coli

It has been shown that the synthesis of an export-defective protein can interfere with the normal export process in Escherichia coli by limiting the availability of SecB protein, a component of the export apparatus (Collier, D.N., Bankaitis, V.A., Weiss, J.B., and Bassford, P.J. (1988) Cell 53, 273-283). Consistent with this observation, we find that the interference elicited by an export-defective LamB protein is a titratable response resulting from the limitation of a single ligand. We have mapped the interfering region in LamB to between amino acids 320 and 380 of the mature protein. Expression of this sequence in the form of a LacZ-LamB-LacZ fusion protein elicits the export interference phenotype. Deletion of the sequence from an export-defective LamB protein eliminates the ability of this protein to interfere with the export of other secreted proteins. Together, these findings show that this sequence is both necessary and sufficient to cause export interference. Surprisingly, deletion of this sequence from an otherwise wild-type LamB protein does not cause the mutant LamB product to exhibit any obvious export defect. Based on our results, we propose that SecB interacts with both amino acids 320-380 of mature LamB and the LamB signal sequence during initiation of the export process.     

The presence of both the signal sequence and a region of mature LamB protein is required for the interaction of LamB with the export factor SecB

In the accompanying paper (Altman, E., Bankaitis, V.A., and Emr, S.D. (1990) J. Biol. Chem. 265, 18148-18153) a putative SecB binding site was identified in the mature LamB protein. The export of wild-type LamB was unperturbed when this region was removed, however, suggesting the presence of a second site of interaction between SecB and LamB. In this paper we show that the interference caused by export-defective LamB proteins is influenced by the amount of signal sequence that is present. If a large portion of the signal sequence is deleted then the interference levels are significantly reduced. This result suggests that a region of the signal sequence contributes to the interaction of SecB with the LamB protein. Using anti-SecB affinity chromatography, we demonstrated directly that the association of SecB protein with precursor LamB is dependent on the presence of both the LamB signal sequence and the interfering region which maps to amino acids 320-380 of mature LamB. Although the interfering region is not necessary for the export of wild-type LamB under normal conditions, when the signal sequence is mutationally altered the interfering region is required to promote the efficient export of LamB protein. Also, deletion of the interfering region eliminates the ability of wild-type LamB precursor to be maintained in an export competent conformation in vivo. Collectively, our results indicate that efficient export of the LamB protein is achieved by an interaction with SecB that involves both the LamB signal sequence and the interfering region in mature LamB.     

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.     

Signal sequence mutations as tools for the characterization of LamB folding intermediates

lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequence processing. We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within the LamB signal sequence. Most interesting were mutations that changed an amino acid with a strong positive charge to an amino acid with no charge. Further characterization of two such suppressors revealed that they produce functional LamB that is localized to the outer membrane with its entire signal sequence still attached. Biochemical analysis shows that mutant LamB monomer chases into an oligomeric species with properties different from those of wild-type LamB trimer. Because assembly of mutant LamB is slowed, these mutations provide useful tools for the characterization of LamB folding intermediates.     

Signal sequence mutations that alter coupling of secretion and translation of an Escherichia coli outer membrane protein.

The lamB701-708 signal sequence mutation reduces expression of LamB, an outer membrane protein of Escherichia coli. To investigate the possibility that synthesis and export of LamB are coupled, as suggested by the expression defect of the lamB701-708 mutation, we isolated intragenic suppressors of the lamB701-708 mutation. The expression defect imposed by the lamB701-708 mutation is suppressed by an export-defective signal sequence mutation, suggesting that translation and export are coupled. The additional observation that not all export-defective signal sequence mutations suppressed the lamB701-708 expression defect suggests that translational arrest can be uncoupled from export.     

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 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.     

Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12

It has been proposed that the efficient localization of the outer membrane protein LamB requires a functional signal sequence and at least two additional regions contained within the mature protein. We define these regions more precisely by deletion analysis, and we describe methods for cloning deleterious lacZ fusions onto high-copy-number plasmids and generating in-frame deletions. Analysis of the effects of a series of internal lamB deletions on the export of a LamB-LacZ hybrid protein and of the LamB protein itself indicates that necessary informational signal(s) required for localization lie at the amino-terminal end of the protein. In addition, our analysis indicates that there is a region of information close to or within the fusion joint of the largest lamB-lacZ fusion that increases the efficiency of the export process. A unique deletion that removes a protein segment from amino acid 70 to 200 appears to prevent proteolytic removal of the signal sequence. Nevertheless, the mutant protein is exported to the outer membrane.     

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.     

Kinetic analysis of lamB mutants suggests the signal sequence plays multiple roles in protein export

We have developed a quantitative assay to measure the rate of processing of precursor LamB into mature protein and have used this assay to characterize 10 previously isolated and 3 new lamB signal sequence mutants. The data suggest that the LamB signal sequence serves a complex function. Our assay has revealed five types of signal sequence defect: 1) a strong kinetic defect resulting from alteration of the secondary structure in the putative alpha-helical region in the hydrophobic core, 2) a strong, or 3) a weak kinetic defect due to placement of a charged residue in the hydrophobic core, 4) decreased synthesis of LamB, and 5) both a decrease in synthesis and a strong kinetic defect. The effect of an extragenic suppressor, prlA4 on the rate of processing pLamB containing signal sequence mutations was also examined and compared to the rates in wild-type strains. It was found that prlA4 increases the rate of processing in some, but not all, mutants having a kinetic defect while having no effect on the decreased synthesis seen in mutants of types 4 and 5.     

Translational control of exported proteins that results from OmpC porin overexpression.

The regulation of synthesis and export of outer membrane proteins of Escherichia coli was examined by overexpressing ompC in multicopy either from its own promoter or from an inducible promoter in an expression vector. Overexpression of OmpC protein resulted in a nearly complete inhibition of synthesis of the OmpA and LamB outer membrane proteins but had no effect on synthesis of the periplasmic maltose-binding protein. Immunoprecipitation of labeled proteins showed no evidence of accumulation of uncleaved precursor forms of OmpA or maltose-binding protein following induction of OmpC overexpression. The inhibition of OmpA and LamB was tightly coupled to OmpC overexpression and occurred very rapidly, reaching a high level within 2 min after induction. OmpC overexpression did not cause a significant decrease in expression of a LamB-LacZ hybrid protein produced from a lamB-lacZ fusion in which the fusion joint was at the second amino acid of the LamB signal sequence. There was no significant decrease in rate of synthesis of ompA mRNA as measured by filter hybridization of pulse-labeled RNA. These results indicate that the inhibition is at the level of translation. We propose that cells are able to monitor expression of exported proteins by sensing occupancy of some limiting component in the export machinery and use this to regulate translation of these proteins.     

Linker mutagenesis in the gene of an outer membrane protein of Escherichia coli, lamB

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in-phase deletions or small insertions. Except for two in-phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.     

Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli

We describe a lesion, lamB701-708, affecting the hydrophilic portion of the lambda receptor signal sequence. The C to A transversion of the sixth codon of the signal sequence changes a positively charged arginine to a neutral serine. The phenotype conferred by this alteration is unique among previously described signal sequence mutations. The results suggest an essential role for the charged amino acids of the hydrophilic segment in the initial interaction between a nascent secreted protein and a membrane export site. The results further suggest that synthesis of lambda receptor is coupled to its export.     

A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation.

We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein. The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB. It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan. Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional. High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid. By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations. Many of the mutations decrease expression of the TrpC-TonB protein, as expected. In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB. The mutant proteins are designated TrpC-TonBG26D. The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations. TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm. We conclude that the Gly----Asp substitution is an export mutation. TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein.     

Mutational alterations affecting the export competence of a truncated but fully functional maltose-binding protein signal peptide.

The wild-type maltose-binding protein (MBP) signal peptide is 26 amino acids in length. A mutationally altered MBP signal peptide has been previously described that is missing one of the basic residues from the hydrophilic segment and seven residues from the hydrophobic core; however, it still facilitates MBP secretion to the periplasm at a rate and efficiency comparable to those of the wild-type structure. Thus, this truncated signal peptide (designated the R2 signal peptide) must retain all of the essential features required for proper export function. In this study, alterations were obtained in the R2 signal peptide that resulted in an export-defective MBP. For the first time, signal sequence mutations were obtained that resulted in the synthesis of a totally export-defective MBP. As was previously the case for the wild-type signal peptide, the introduction of either charged residues or helix-breaking proline residues adversely affected export function. Despite these similarities, the position of these alterations within the R2 signal peptide, their relative effects on MBP secretion and processing, and an analysis of the ability of various extragenic prl mutations to suppress the secretion defects provide additional insight into the minimal requirements for a functional MBP signal peptide.     

Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants

Escherichia coli strains harboring malE signal sequence point mutations accumulate export-defective precursor maltose-binding protein (MBP) in the cytoplasm. Beginning with these mutants, a number of spontaneous intragenic revertants have been obtained in which export of the MBP to the periplasm is either partially or totally restored. With a single exception, each of the reversion mutations resulted in an increase in the overall hydrophobicity of the signal peptide hydrophobic core by one of five different mechanisms. In some revertants, MBP export was achieved at a rate comparable to the wild type MBP; in other cases, the rate of MBP export was significantly slower than wild type. The results indicate that the overall hydrophobicity of the signal peptide, rather than the absolute length of its uninterrupted hydrophobic core, is a major determinant of MBP export competency. An alteration at residue 19 of the mature MBP also has been identified that provides fairly efficient suppression of the export defect in the adjacent signal peptide, further suggesting that important export information may reside in this region of the precursor protein.     

Characterization of the type III export signal of the flagellar hook scaffolding protein FlgD of Escherichia coli

Transport of flagellar structural proteins beyond the cytoplasmic membrane is accomplished by a type III secretory pathway [flagellar type III secretion system (fTTSS)]. The mechanism of substrate recognition by the fTTSS is still enigmatic. Using the hook scaffolding protein FlgD of Escherichia coli as a model substrate, it is demonstrated that the export signal is contained within the N-terminal 71 amino acids of FlgD. Analysis of frame-shift mutations and alterations of the nucleotide sequence suggest a proteinaceous nature of the signal. Furthermore, the physicochemical properties of the first about eight amino acids are crucial for export.     

Characterization of two isoforms of the skeletal muscle LIM protein 1, SLIM1. Localization of SLIM1 at focal adhesions and the isoform slimmer in the nucleus of myoblasts and cytoplasm of myotubes suggests distinct roles in the cytoskeleton and in nuclear-cytoplasmic communication.

We have cloned and characterized a novel isoform of the skeletal muscle LIM protein 1 (SLIM1), designated SLIMMER. SLIM1 contains an N-terminal single zinc finger followed by four LIM domains. SLIMMER is identical to SLIM1 over the first three LIM domains but contains a novel C-terminal 96 amino acids with three potential bipartite nuclear localization signals, a putative nuclear export sequence, and 27 amino acids identical to the RBP-J binding region of KyoT2, a murine isoform of SLIM1. SLIM1 localized to the cytosol of Sol8 myoblasts and myotubes. SLIMMER was detected in the nucleus of myoblasts and, following differentiation into myotubes, was exclusively cytosolic. Recombinant green fluorescent protein-SLIM1 localized to the cytoplasm and associated with focal adhesions and actin filaments in COS-7 cells, while green fluorescent protein-SLIMMER was predominantly nuclear. SLIMMER truncation mutants revealed that the first nuclear localization signal mediates nuclear localization. The addition of the proposed nuclear export sequence decreased the level of exclusively nuclear expression and increased cytosolic SLIMMER expression in COS-7 cells. The leucine-rich nuclear export signal was required for the export of SLIMMER from the nucleus of myoblasts to the cytoplasm of myotubes. Collectively, these results suggest distinct roles for SLIM1 and SLIMMER in focal adhesions and nuclear-cytoplasmic communication.     

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.     

Mutagenesis by random linker insertion into the lamB gene of Escherichia coli K12

Summary Gene lamB encodes an outer membrane protein involved in maltose and maltodextrin transport as well as phage adsorption. The active form is a trimer. We characterized 11 mutations in lamB, obtained after random insertion of a BamH1 linker and screening for stable immunodetectable mutant proteins. Six mutations resulted in the loss of the distal part of the LamB protein either by deletion (five cases) or frameshift (one case). The six corresponding proteins had all lost the ability to confer phage sensitivity and the capacity to grow on dextrins, and to yield immunnodetectable oligomers. Induction of a high level of the four longest of these proteins was toxic to the cell. Five other mutations were due to in-frame insertions. In four cases, the corresponding proteins still had the ability to yield immunodetectable oligomers, to confer phage sensitivity and the capacity to grow on dextrins and were not toxic on induction. In one case (AJC73), the mutant protein had lost the first three properties and was toxic on induction. Deletions and duplications between some of the inserts were also constructed and studied. To account for our results we present a hypothetical scheme in which trimerization would not only be needed for phage sensitivity and growth on dextrins but also for proper insertion into the outer membrane. The C-terminus of the protein, as well as other regions such as the site of mutation AJC73, would be required for the formation of stable trimers. We tentatively interpret toxicity as due to improper insertion into the outer membrane. Our results also show that it is possible to insert several amino acids (up to 11 in one case) at a number of positions in LamB without appreciably affecting its export and activities.