SipY Is the Streptomyces lividans type I signal peptidase exerting a major effect on protein secretion

Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins. The developmental complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (sipW, sipX, sipY, and sipZ) unusually clustered in its chromosome. Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual sip mutants. None of the sip genes seemed to be essential for bacterial growth. Analysis of total extracellular proteins indicated that SipY is likely to be the major S. lividans SPase, since the sipY mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.     

Functional analysis of the Streptomyces lividans type I signal peptidases

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.     

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.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.     

Membrane topology of the Streptomyces lividans type I signal peptidases

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.     

Peptidases affecting recombinant protein production by Streptomyces lividans

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.     

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.     

The Streptomyces lividans cytoplasmic signal recognition particle receptor FtsY is involved in protein secretion

The bacterial version of the mammalian signal recognition particle (SRP) and its receptor alpha-subunit (FtsY) is well conserved and essential to all known bacteria. In gram-negative bacteria, the SRP pathway mediates a co-translational targeting of most inner membrane proteins. Additionally, in Streptomyces lividans, a gram-positive bacterium, SRP also targets secretory proteins to the translocon. The role of S. lividans FtsY has been assessed in this work. Co-immunoprecipitation studies confirmed that FtsY is associated with the S. lividans SRP in the cytoplasm and that this complex also co-immunoprecipitated with pre-agarase, suggesting that the SRP receptor is involved in SRP-mediated targeting of secretory proteins in S. lividans. Furthermore, the SRP remains attached for the most part to the cellular membrane when the cleavage of pre-secretory proteins is severely reduced in a strain lacking the gene coding for the major type-I signal peptidase.     

Factors governing nonoverlapping substrate specificity by mitochondrial inner membrane peptidase

At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins. One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae. Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities. A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the -1 and -3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides. We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position. We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p. A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases.     

A role for type I signal peptidase in Staphylococcus aureus quorum sensing

The Staphylococcus aureus Agr quorum-sensing system modulates the expression of extracellular virulence factors. The Agr system is controlled by an autoinducing peptide (AIP) molecule that is secreted during growth. In the AIP biosynthetic pathway, two proteolytic events are required to remove the leader and tail segments of AgrD, the peptide precursor of AIP. The only protein known to be involved in this pathway is AgrB, a membrane endopeptidase that removes the AgrD carboxy-tail. We designed a synthetic peptide substrate and developed an assay to detect peptidases that can remove the N-terminal leader of AIP. Several peptidase activities were detected in S. aureus extracts and these activities were present in both wild-type and agr mutant strains. Only one of these peptidases cleaved in the correct position and all properties of this enzyme were consistent with type I signal peptidase. Subsequent cloning and purification of the two known S. aureus signal peptidases, SpsA and SpsB, demonstrated that only SpsB catalysed this activity in vitro. To investigate the role of SpsB in AIP biosynthesis, SpsB peptide inhibitors were designed and characterized. The most effective inhibitor blocked SpsB activity in vitro and showed antibacterial activity against S. aureus. Importantly, the inhibitor reduced expression of an Agr-dependent reporter and inhibited AIP production in S. aureus, indicating a role for SpsB in quorum sensing.     

Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.     

Genetic complementation in yeast reveals functional similarities between the catalytic subunits of mammalian signal peptidase complex

Type I signal peptidases (SPs) comprise a family of structurally related enzymes that cleave signal peptides from precursor proteins following their transport out of the cytoplasmic space in eukaryotic and prokaryotic cells. One such enzyme, the mitochondrial inner membrane peptidase, has two catalytic subunits, which recognize distinct cleavage site motifs in their signal peptide substrates. The only other known type I SP with two catalytic subunits is the signal peptidase complex (SPC) in the mammalian endoplasmic reticulum. Here, we tested the hypothesis that, as with inner membrane peptidase catalytic subunits, SPC catalytic subunits exhibit nonoverlapping substrate specificity. We constructed two yeast strains without endogenous SP, one expressing canine SPC18 and the other expressing a truncation of canine SPC21 (SPC21 Delta N), which lacks 24 N-terminal residues that prevent expression of SPC21 in yeast. By monitoring a variety of soluble and membrane-bound substrates, we find that, in contrast to the tested hypothesis, SPC catalytic subunits exhibit overlapping substrate specificity. SPC18 and SPC21 Delta N do, however, cleave some substrates with different efficiencies, although no pattern for this behavior could be discerned. In light of the functional similarities between SPC proteins, we developed a membrane protein fragmentation assay to monitor the position of the catalytic sites relative to the surface of the endoplasmic reticulum membrane. Using this assay, our results suggest that the active sites of SPC18 and SPC21 Delta N are located 4-11 A above the membrane surface. These data, thus, support a model that SPC18 and SPC21 are functionally and structurally similar to each other.     

Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus.

The gene, spsB, encoding a type I signal peptidase has been cloned from the gram-positive eubacterium Staphylococcus aureus. The gene encodes a protein of 191 amino acid residues with a calculated molecular mass of 21,692 Da. Comparison of the protein sequence with those of known type I signal peptidases indicates conservation of amino acid residues known to be important or essential for catalytic activity. The enzyme has been expressed to high levels in Escherichia coli and has been demonstrated to possess enzymatic activity against E. coli preproteins in vivo. Experiments whereby the spsB gene was transferred to a plasmid that is temperature sensitive for replication indicate that spsB is an essential gene. We identified an open reading frame immediately upstream of the spsB gene which encodes a type I signal peptidase homolog of 174 amino acid residues with a calculated molecular mass of 20,146 Da that is predicted to be devoid of catalytic activity.     

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.     

Expression of the Bacillus subtilis TasA signal peptide leads to cell death in Escherichia coli due to inefficient cleavage by LepB

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2′) to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2′, inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.     

The role of the conserved box E residues in the active site of the Escherichia coli type I signal peptidase

Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins. These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases. Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme. First, we find that there is no requirement for activity for a salt bridge between the invariant Asp-273 and the Arg-146 residues. In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserved salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential for signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms. We propose that replacement of the hydrogen side chain of Gly-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280.     

The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains

The Gram-positive eubacterium Streptomyces lividans contains four chromosomally encoded type I signal peptidases, SipW, SipX, SipY and SipZ, of which all but SipW have an unusual C-terminal membrane anchor. For in vitro characterisation of these signal peptidases, the S. lividans sip genes were expressed in Escherichia coli and the corresponding proteins were purified. The four enzymes had an optimum activity at an alkaline pH, notably pH 8-9 for SipW and SipY and pH 10-11 for SipX and SipZ. In contrast to SipW, the in vitro activities of SipX, SipY and SipZ significantly increased in the presence of detergent. Since none of the S. lividans Sip proteins contains the hydrophobic beta-barrel domain, which in E. coli LepB was proven to be requisite for detergent-dependent in vitro activity, we assume that for detergent dependence, the C-terminal transmembrane anchor can partly substitute for this domain. Finally, all Sip proteins were stimulated by added phospholipids, which strongly suggests that phospholipids play an important role in the catalytic mechanism.     

Biochemical characterization of signal peptidase I from gram-positive Streptococcus pneumoniae

Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases and Escherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.