Cloning and expression of a gene coding for the prolipoprotein signal peptidase of Escherichia coli

An Escherichia coli mutant, Y815, has a temperature-sensitive prolipoprotein signal peptidase. IPTG-induced synthesis of the major outer membrane prolipoprotein (PLP) results in the inhibition of cell growth because of accumulation of PLP in its envelope [J. Bacteriol. (1982) 152, 1163-1168]. The 2000 E. coli strains of Clarke and Carbon's collection were screened for the presence of a plasmid complementing the IPTG-sensitivity of the growth of Y815. One plasmid, pLC3-13, complemented the IPTG-sensitivity. The envelope fraction prepared from Y815 transformed by pLC3-13 showed high activity of the PLP signal peptidase in vitro at high temperature. A 4 kb AccI fragment subcloned onto plasmid pHY001 was shown to carry the gene for the PLP signal peptidase.     

Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage

1. Download : Download high-res image (350KB)
2. Download : Download full-size image

     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Genetic characterization of a gene for prolipoprotein signal peptidase in Escherichia coli

A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.     

The chemistry and enzymology of the type I signal peptidases

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.     

Plastidic type I signal peptidase 1 is a redox‐dependent thylakoidal processing peptidase

Thylakoids are the photosynthetic membranes in chloroplasts and cyanobacteria. The aqueous phase inside the thylakoid known as the thylakoid lumen plays an essential role in the photosynthetic electron transport. The presence and significance of thiol‐disulfide exchange in this compartment have been recognized but remain poorly understood. All proteins found free in the thylakoid lumen and some proteins associated to the thylakoid membrane require an N‐terminal targeting signal, which is removed in the lumen by a membrane‐bound serine protease called thylakoidal processing peptidase (TPP). TPP is homologous to Escherichia coli type I signal peptidase (SPI) called LepB. Genetic data indicate that plastidic SPI 1 (Plsp1) is the main TPP in Arabidopsis thaliana (Arabidopsis) although biochemical evidence had been lacking. Here we demonstrate catalytic activity of bacterially produced Arabidopsis Plsp1. Recombinant Plsp1 showed processing activity against various TPP substrates at a level comparable to that of LepB. Plsp1 and LepB were also similar in the pH optima, sensitivity to arylomycin variants and a preference for the residue at ?3 to the cleavage site within a substrate. Plsp1 orthologs found in angiosperms contain two unique Cys residues located in the lumen. Results of processing assays suggested that these residues were redox active and formation of a disulfide bond between them was necessary for the activity of recombinant Arabidopsis Plsp1. Furthermore, Plsp1 in Arabidopsis and pea thylakoids migrated faster under non‐reducing conditions than under reducing conditions on SDS‐PAGE. These results underpin the notion that Plsp1 is a redox‐dependent signal peptidase in the thylakoid lumen.     

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b5 precursor is an exocytoplasmic post-translocational event in Escherichia coli

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b₅ precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.     

Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli.

The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C-terminal deletions or fusions of the livK gene to trpA (encoding the alpha subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the alpha-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.     

Identification of residues essential for the catalytic activity of Sec11b, one of the two type I signal peptidases of Haloferax volcanii

Sec11b is one of two signal peptidases (SPases) in the haloarchaeon Haloferax volcanii. Site-directed mutagenesis revealed Ser-72, His-137 and Asp-187 as essential for signal peptide cleavage. Thus, like the SPase of the methanoarchaeon Methanococcus voltae, H. volcanii Sec11b uses a catalytic mechanism reminiscent of its eukaryal rather than its bacterial counterpart. The availability of an additional model system to study the archaeal SPase, now in the form of the purified protein, promises additional insight into the behavior of this enzyme.     

Analysis of type I signal peptidase affinity and specificity for preprotein substrates

Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins. Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance. The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis. In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M). The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity. The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening. In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity.     

Structure and mechanism of Escherichia coli type I signal peptidase

Type I signal peptidase is the enzyme responsible for cleaving off the amino-terminal signal peptide from proteins that are secreted across the bacterial cytoplasmic membrane. It is an essential membrane bound enzyme whose serine/lysine catalytic dyad resides on the exo-cytoplasmic surface of the bacterial membrane. This review discusses the progress that has been made in the structural and mechanistic characterization of Escherichia coli type I signal peptidase (SPase I) as well as efforts to develop a novel class of antibiotics based on SPase I inhibition. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Coexpression of the type I signal peptidase gene sipM increases recombinant protein production and export in Bacillus megaterium MS941

The removal of the signal peptide from a precursor protein is a crucial step of protein secretion. In order to improve Bacillus megaterium as protein production and secretion host, the influence of homologous type I signal peptidase SipM overproduction on recombinant Leuconostoc mesenteroides dextransucrase DsrS synthesis and export was investigated. The dsrS gene was integrated as a single copy into the chromosomal bgaM locus encoding beta-galactosidase. Desired clones were identified by blue-white selection. In this strain, the expression of sipM from a multicopy plasmid using its own promoter increased the amount of secreted DsrS 3.7-fold. This increase in protein secretion by SipM overproduction was next transferred to a high level DsrS production strain using a multicopy plasmid encoding sipM with its natural promoter and dsrS under control of a strong xylose-inducible promoter. No further increase in DsrS export were observed when this vector was carrying two sipM copies. Similarly, bicistronic sipM and dsrS high level expression did not enhance DsrS secretion, indicating the natural limitation of the approach. Interestingly, SipM-enhanced DsrS secretion also resulted in an overall increase of DsrS production.     

Signal peptidase I: cleaving the way to mature proteins

Signal peptidase I (SPase I) is critical for the release of translocated preproteins from the membrane as they are transported from a cytoplasmic site of synthesis to extracytoplasmic locations. These proteins are synthesized with an amino-terminal extension, the signal sequence, which directs the preprotein to the Sec- or Tat-translocation pathway. Recent evidence indicates that the SPase I cleaves preproteins as they emerge from either pathway, though the steps involved are unclear. Now that the structure of many translocation pathway components has been elucidated, it is critical to determine how these components work in concert to support protein translocation and cleavage. Molecular modeling and NMR studies have provided insight on how the preprotein docks on SPase I in preparation for cleavage. This is a key area for future work since SPase I enzymes in a variety of species have now been identified and the inhibition of these enzymes by antibiotics is being pursued. The eubacterial SPase I is essential for cell viability and belongs to a unique group of serine endoproteases which utilize a Ser-Lys catalytic dyad instead of the prototypical Ser-His-Asp triad used by eukaryotes. As such, SPase I is a desirable antimicrobial target. Advances in our understanding of how the preprotein interfaces with SPase I during the final stages of translocation will facilitate future development of inhibitors that display a high efficacy against SPase I function.     

A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc₁ complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the F_Ad subunit of the ATP synthetase and the tobacco F₁ subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bc₁ complex of the respiratory chain.     

Induction of ethanol dependence increases signal peptidase mRNA levels in rat brain

Differential Northern blot hybridization was used as a screening tool to identify mRNAs that respond quantitatively to the induction of ethanol dependence. Adult male rats were treated with repeated, high doses of ethanol for 4 consecutive days. This regimen resulted in the development of tolerance and dependence upon ethanol. RNA isolated from the ethanol-dependent rat brains was used to construct a cDNA library. One cDNA was identified that hybridized to a mRNA which increased in rat brain during the ethanol treatment. Sequence analysis of the cDNA indicated that it recognized a mRNA in rat brain which was very similar to that which encodes the 18 kDa subunit of canine signal peptidase. The rat signal peptidase mRNA was observed to increase in brain nearly 2-fold within 48 h after the initiation of ethanol treatment. Ethanol did not significantly alter -actin mRNA levels during the treatment period. These results support the existence of an ethanol-responsive signal peptidase mRNA in rat brain.     

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.     

Cloning and sequence analysis of a signal peptidase I from the thermophilic cyanobacterium Phormidium laminosum

Type I signal peptidases are a widespread family of enzymes which remove the presequences from proteins translocated across cell membranes, including thylakoid and cytoplasmic membranes of cyanobacteria and thylakoid membranes of chloroplasts. We have cloned and sequenced a signal peptidase gene from the thermophilic cyanobacterium Phormidium laminosum which is believed to encode an enzyme common to both membrane systems. The deduced amino acid sequence is 203 residues long and although the overall similarity among signal peptidases is rather low there are a number of identifiable conserved regions present. The P. laminosum enzyme is predicted to have a single transmembrane domain, in contrast to other Gram-negative bacterial sequences, but similar to other type I signal peptidases.     

Expression study with the Escherichia coli lep gene for leader peptidase in phototrophic purple bacteria

Synthesis and assembly of leader peptidase of Escherichia coli (signal peptidase I), was studied by heterologous expression of its lep gene in three species of phototrophic purple bacteria. Cell extracts of the recipient species showed neither cross reaction with antibodies against E. coli leader peptidase nor cleavage of the model substrate M13-procoat in vitro. The lep gene was transferred via conjugation using the plasmid expression vector for phototrophic bacteria pJAJ9. Plasmidborne leader peptidase enzyme was identified by immunochemical means. However, extracts of transconjugant cells showed no cleavage function. Trypsin digestion studies revealed that the enzyme was not properly integrated across the host membranes. The data suggest that cleaving enzymes for protein export and/or their assembly pathway in purple bacteria differ from the E. coli type.Abbreviations DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate