Functional limits of conformation, hydrophobicity, and steric constraints in prokaryotic signal peptide cleavage regions. Wild type transport by a simple polymeric signal sequence

These experiments examine the role of conformation, hydrophobicity, and steric constraints in the function of the prokaryotic signal peptide cleavage region. The experimental strategy involves replacement of the wild type Escherichia coli alkaline phosphatase signal peptide cleavage region with a series of idealized model sequences designed to epitomize the particular structural and physical variables under study. By analyzing model sequences whose conformations have been determined by physical studies, we have demonstrated that efficient transport does not depend on the structural preference of the cleavage region. Although previous studies based on Chou-Fasman analysis have suggested that the cleavage region forms a beta-turn which is required for transport, our results demonstrate that either a beta-turn- or alpha-helix-fostering sequence in the cleavage region functions indistinguishably from wild type. Furthermore, the presence of a proline residue between the core and cleavage region, although common in natural sequences, is not essential for export. Cleavage regions of varying hydrophobicities can support translocation across the inner membrane, but the placement of bulky residues at positions -1 and -3 upstream of the cleavage site abolishes processing and transport to the periplasm. By reducing the signal peptide to simplified, idealized segments, this study has identified a largely polymeric sequence, MKQST(L10)-(A6), that functions equivalently to the wild type alkaline phosphatase signal peptide. This work starts to provide a basis for the design of a universal prokaryotic signal peptide that incorporates all the critical physical and structural characteristics required for transport function.     

Plastid-targeting peptides from the chlorarachniophyte Bigelowiella natans

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.     

Different sequence patterns in signal peptides from mycoplasmas, other gram-positive bacteria, and Escherichia coli: a multivariate data analysis

Signal peptides are essential N-terminal extensions in export proteins, and have a positively charged N-terminus, a hydrophobic central core, and a C-terminal cleavage region. They interact in a consecutive manner with different accessory proteins during the secretion process. Potential patterns or periodicity in the amino acid (aa) sequence were searched, using multivariate techniques, for a large number of signal peptides from mollicutes (mycoplasmas), other Gram-positive bacteria, and Escherichia coli. Mollicutes signal peptides were significantly different from the E. coli and Gram-positive ones by their N-terminal charge, peptide length, and especially, unique periodicities of side chain hydrophobicity and volumes. Their lipoprotein signal peptides were longer than for any other bacteria. Significant differences were also recorded between the other bacterial peptide groups. Specific aa patterns were more related within the signal peptides from several groups of secreted bacillus enzymes, than for all signal peptides from one bacillus species. In E. coli, signal peptides from proteins routed for the various destinations revealed significant and compartment-specific sequence patterns not evident by other methods. This was substantiated from a large number of signal peptide secretion mutants for the E. coli periplasmic space. It is proposed that the differences in aa patterns and side-chain properties are related to the secondary structure sidedness and topology of the signal peptides, and important for specific interactions during the secretion process.     

Efficiency of erythropoietin's signal peptide for HIV(MN)-1 gp 120 expression

The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used.     

Juxtaposition of signal-peptide charge and core region hydrophobicity is critical for functional signal peptides

In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.     

Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs

Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins. Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity. In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo. A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core. These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase. The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time. The structures obtained were correlated with in vitro bioactivities. Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers. The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent. The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides. The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay.     

Feature-based reappraisal of the Bacillus subtilis exoproteome

Proteomics-based verification of computer-assisted predictions on bacterial protein export have indicated that problems occur with the distinction between (Sec-type) signal peptides that govern protein secretion, and lipoprotein signal peptides or amino-terminal membrane anchors that cause protein retention in the membrane. Therefore, the main aim of this study was to investigate whether feature-based predictions by the SecretomeP (SecP) algorithm will aid the proteomics-based analysis of protein export in Bacillus subtilis. The SecP algorithm is trained to recognize features such as secondary structure and disordered regions, which are generally present in secreted proteins. The results showed that membrane-retained proteins receive, in general, high SecP scores, similar to the scores of secretory proteins. Importantly, the SecP algorithm aided in the re-evaluation of a class of previously identified proteins that remain attached to the membrane despite the presence of an apparent Sec-type signal peptide. These so-called 'Sec-attached' proteins receive on average a lower SecP score, and several of these proteins could be unmasked as transmembrane proteins by combined SecP and signal peptide analyses. Finally, the present study suggests that feature-based outlier analysis may provide leads towards the discovery of novel special-purpose pathways for bacterial protein export.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

Site-directed mutagenesis of the hydrogenase signal peptide consensus box prevents export of a beta-lactamase fusion protein.

A secretion vector, pVN1, expressing the [NiFe] hydrogenase signal peptide of Desulfovibrio vulgaris Hildenborough fused to beta-lactamase from Escherichia coli was constructed in order to study the unusual characteristics of hydrogenase signal peptides, which share a strictly conserved sequence, the consensus box: R-R-X-F-X-K. Although the hydrogenase signal peptide-beta-lactamase fusion protein was processed much more slowly than the fusion of beta-lactamase with its own signal peptide, the system mimicked several features expected for hydrogenase biosynthesis in E. coli, including increased export under anaerobic conditions. Site-directed mutagenesis of R(-28), the first arginine residue of the consensus box, to a glutamate completely inhibited export and processing of the fusion protein. The same mutation of R(-33), located outside the consensus box, had almost no effect. The data indicate a specific role for the consensus box sequence in the export mechanism for hydrogenase.     

A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life

We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.     

Identification of a Post-targeting Step Required for Efficient Cotranslational Translocation of Proteins across the Escherichia coli Inner Membrane

Recent studies have shown that cytoplasmic proteins are exported efficiently in Escherichia coli only if they are attached to signal peptides that are recognized by the signal recognition particle and are thereby targeted to the SecYEG complex cotranslationally. The evidence suggests that the entry of these proteins into the secretory pathway at an early stage of translation is necessary to prevent them from folding into a translocation-incompetent conformation. We found, however, that several glycolytic enzymes attached to signal peptides that are recognized by the signal recognition particle were exported inefficiently. Based on previous studies of post-translational export, we hypothesized that the export block was due to the presence of basic residues at the extreme N terminus of each enzyme. Consistent with our hypothesis, we found that the introduction of negatively charged residues into this segment increased the efficiency of export. Export efficiency was sensitive to the number, position, and sequence context of charged residues. The importance of charge for efficient export was underscored by an in silico analysis that revealed a conserved negative charge bias at the N terminus of the mature region of bacterial presecretory proteins. Our results demonstrate that cotranslational targeting of a protein to the E. coli SecYEG complex does not ensure its export but that export also depends on a subsequent event (most likely the initiation of translocation) that involves sequences both within and just beyond the signal peptide.Since the “signal hypothesis” was proposed over 30 years ago (1), it has become clear that signal sequences are not simply generic hydrophobic peptides that earmark proteins for secretion. In bacteria, the features of a signal peptide determine the mechanism by which a given presecretory protein is targeted to the SecYEG translocation complex in the inner membrane (IM).² Whereas most or all signal peptides are recognized by the signal recognition particle (SRP) in mammalian cells, only a small fraction of Escherichia coli signal peptides are recognized by SRP. These signal peptides are typically extremely hydrophobic (2, 3), but SRP apparently can also recognize slightly less hydrophobic signal peptides that contain a highly basic N terminus (4). SRP recognizes signal peptides as they emerge from translating ribosomes and then targets ribosome-nascent chain complexes to the IM cotranslationally (5). The binding of SRP to its receptor (FtsY), which interacts with the SecYEG complex (6), leads to the release of the nascent chain in the immediate vicinity of the translocation machinery. By targeting nascent polypeptides to the SecYEG complex at an early stage of translation, SRP prevents its substrates from folding into a conformation that is incompatible with translocation through the narrow channel formed by the SecYEG complex (7). Because most signal peptides are not recognized by E. coli SRP, the majority of presecretory proteins are fully synthesized and targeted post-translationally to the IM. These proteins are maintained in a translocation-competent conformation by molecular chaperones such as SecB that keep them unfolded (or loosely folded) (8). Signal peptides themselves also appear to play a role in maintaining translocation competence (9, 10). After mediating the targeting reaction, signal peptides likely play a role in gating open the SecYEG complex to initiate translocation.Interestingly, although signal sequences are the most salient feature of presecretory proteins, they are neither completely necessary nor sufficient to mediate protein export in E. coli (11–13). A version of alkaline phosphatase that lacks a signal peptide is still exported, albeit very inefficiently (11). The export of the leaderless protein, unlike the export of wild-type alkaline phosphatase, is strictly dependent on SecB (11). Conversely, the attachment of signal peptides to cytoplasmic proteins often does not promote their export (14). In light of evidence that folding and export are competing events, these observations led to the proposal that exported proteins tend to fold slowly (or are prevented from folding by chaperones) and therefore remain translocation-competent even without a signal peptide, whereas cytoplasmic proteins fold rapidly into a conformation that is incompatible with export. Recent studies that used thioredoxin as a model protein have validated this hypothesis. Whereas the wild-type protein attached to a typical signal peptide remained trapped in the cytoplasm, four of five slow folding mutants were exported efficiently (15). Furthermore, attachment of a signal peptide that is recognized by SRP to thioredoxin led to efficient export (16). This idea was further confirmed by a report in which various DARPins (designed ankyrin Repeat proteins) were attached to different signal peptides. Most of the DARPins were exported efficiently when they were fused to signal peptides that mediate cotranslational targeting but remained in the cytoplasm when they were attached to signal peptides that are bypassed by SRP (17).Despite these observations, there are several lines of evidence suggesting that export efficiency is not simply dictated by the ability of a protein to reach the SecYEG complex before folding into a translocation-incompetent conformation. For reasons that are unclear, some DARPins are secreted inefficiently even when they are routed into the SRP pathway (17). In addition, numerous reports have indicated that the amino acid composition of the segment of post-translationally targeted presecretory proteins that lies just beyond the signal peptide cleavage site has a dramatic effect on export efficiency. Statistical analysis has shown that the first ∼5–15 residues of the mature region of most presecretory proteins produced by Gram-negative bacteria is neutral or has a net negative charge (18). Consistent with the observed sequence bias, the presence of multiple basic residues at the N terminus of the mature region often leads to accumulation of the secretory precursor, whereas conversion of the basic residues to acidic residues restores export (19–22). Because different combinations of proteins and signal peptides were used in these studies, the exact number and location of charged residues that impinge on the efficiency of export is unclear. In any case, the effect of the net charge in the region distal to the signal peptide on protein export has never been explained. Although basic residues might conceivably promote premature folding of presecretory proteins or block the cleavage of signal peptides by leader peptidase, it is also possible that they inhibit an uncharacterized post-targeting event. Even if effects on signal peptide cleavage could have been ruled out in the aforementioned studies, however, it would not have been possible to distinguish between effects on protein folding and effects on a hypothetical post-targeting step because only proteins that are targeted post-translationally were monitored.To gain further insight into the factors that govern the efficiency of protein export, we sought an explanation for the observation that the cotranslational targeting of at least some cytoplasmic proteins is insufficient to guarantee their translocation across the IM. We found that the export of several different endogenous E. coli cytoplasmic proteins required not only the attachment of a signal peptide that is recognized by SRP but also a net negative charge just past the signal peptide cleavage site. Taken together with previous results, our data show that the charge of the segment just beyond the signal peptide influences export efficiency irrespective of the mechanism by which a protein is targeted to the IM. Because proteins that are targeted cotranslationally reach the IM before they have a chance to fold, our results imply the existence of a post-targeting step (most likely the initiation of translocation) that is facilitated by acidic residues distal to the signal peptide and inhibited or delayed by basic residues. These results help to resolve a long-standing puzzle about the influence of the mature region of presecretory proteins on protein export and have significant implications for optimizing the export of cytosolic and heterologous proteins in E. coli.     

Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.     

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.     

Fluorescence analysis of tryptophan-containing variants of the LamB signal sequence upon insertion into a lipid bilayer

To investigate the interaction of the LamB signal sequence with lipid bilayers, we have synthesized three tryptophan-containing analogues of the wild-type signal peptide. The tryptophan residues were used as intrinsic fluorescent probes of the N-terminal (position 5), central (position 18), and C-terminal (position 24) regions of the 25-residue peptide. The tryptophan substitutions did not significantly alter the physical properties of the wild-type signal peptide. In the presence of lipid vesicles which mimic the composition of the Escherichia coli inner membrane, the peptides adopt alpha-helical structure, and the tryptophan fluorescence emission maximum is shifted to shorter wavelength, indicating that the peptides insert into the acyl chain region of the lipid bilayer. Fluorescence quenching by soluble, aqueous-phase (I-), and membrane-resident (nitroxide-labeled lipids) quenchers was used to locate the tryptophans in each peptide within the bilayer. The C-terminus was interfacial while the central region of the signal sequence was deeply buried within the acyl chain region of the bilayer. The tryptophan at position 5 was buried but less deeply than the tryptophan at position 18. This topology is consistent with either a looped or a transmembrane orientation of signal peptide. However, either structure must accommodate the high helical content of the peptides in vesicles. These results indicate that the LamB signal sequence spontaneously inserts into the acyl chain region of lipid membranes in the absence of any of the proteins involved in protein secretion.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5′ end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) alpha and interleukin (IL) 10 by fusing the coding sequences of mature hTNFalpha and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the DeltatatB and DeltatatC single mutants, indicating that the transport of hTNFalpha and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFalpha and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the DeltatatB mutant than in the wild-type strain.     

The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.