Use of synthetic signal sequences to explore the protein export machinery

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.     

Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors

Bacterial adherence to and invasion of eukaryotic cells are important mechanisms of pathogenicity. Most Gram-positive bacteria interact with the components of the host extracellular matrix (ECM) to adhere to, colonize and invade cells and tissues. The bacterial proteins that bind to components of the ECM harbour signal sequences for their secretion and mechanisms of anchoring to the host cell surface. However, in recent years, some cell-surface adhesins and invasins of Gram-positive bacteria have been described that do not possess a signal sequence or a membrane anchor. These proteins are secreted by an as-yet-unknown mechanism and are probably localized on the bacterial surface by reassociation. These anchorless but surface-located adhesins and invasins represent a new class of virulence factors.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Signal-sequence Trap in Mammalian and Yeast Cells: A Comparison

Membrane associated and secreted proteins are translated as precursors containing a signal peptide that allows protein-insertion into the membrane of the endoplasmic reticulum and is co-translationally removed in the lumen. The ability of the signal peptide to direct a polypeptide into the secretory pathway is exploited in methods developed to select cDNAs encoding such proteins. Different strategies are known in which cDNA libraries can be screened for signal peptides by the ability of the latter to rescue the translocation of signal sequence-less proteins. In one method, a cDNA library is tested for interleukin 2 receptor α chain translocation to the membrane in COS cells, in another one for invertase secretion from yeast. In this work, we compared the two systems by testing six mouse signal peptides in COS and yeast cells. All of them were functional in the mammalian system, whereas only three of them in yeast. Two other sequences needed the 5′ cDNA sequence flanking the ATG codon to be removed in order to enable protein translocation. Although the structure of signal sequences and the functioning of the secretory machinery are well conserved from prokaryotes to eukaryotes, it seems evident that not all signal peptides can be interchanged between different proteins and organisms. In particular, signal peptides that are functional in the mammalian system do not necessarily lead to protein translocation in yeast. Received: 9 March 2001     

信号肽序列及其在蛋白质表达中的应用

信号肽在蛋白分泌的过程中起重要作用,分泌性蛋白质合成后由信号肽引导其穿过合成所在的细胞到其他组织细胞中。可以利用因特网在线工具和信号序列捕获系统来判定基因序列中是否含有信号肽序列。外源蛋白的表达形式多为细胞内不溶性表达(包涵体),少数为细胞外分泌表达。利用信号肽来引导外源蛋白分泌可避免因包涵体复性带来的困难。研究表明,多种外源基因连接上信号肽后在原核表达系统如大肠杆菌、L型细菌、芽孢杆菌和乳酸杆菌中等都得到了分泌表达;信号肽也广泛应用于真核表达系统如毕赤酵母和昆虫杆状病毒表达系统,以提高蛋白的表达量。     

Estimation of DNA Base Composition by High Performance Liquid Chromatography of Its Nuclease PI Hydrolysate

The Saccharomyces diastaticus glucoamylase encoded by ST A1 contains two signal sequences for potent secretion of the enzyme, a hydrophobic leader peptide (HL), and a tract consisting of threonine- and serine-rich sequences (TS); hybrid proteins of Escherichia coli β-galactosidase carrying both HL and TS are secreted through the cytoplasmic membrane to the cell-surface fraction of yeast cells, but those carrying either HL or TS are not. To investigate the molecular mechanisms for these signal sequences, we have isolated a dominant mutation, SSD1, which suppresses a secretory defect caused by deletion of these sequences. Yeast cells harboring the mutation secreted hybrid β-galactosidase proteins carrying either HL or TS into the cell-surface fraction. Even β-ga!actosidase itself was secreted to the cell surface in the mutant. These results suggest that HL and TS interact with a wild- type ssd1⁺ gene product to promote protein secretion.     

Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors

It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.     

How are the Non-classically Secreted Bacterial Proteins Released into the Extracellular Milieu?

Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.     

Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. We describe here a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III-dependent manner from Yersinia pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first 30 amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. These assays should prove useful to assess the secretion of other chlamydial proteins that are potentially exposed to the cytosol of the host cell.     

Secretion of predicted Inc proteins of Chlamydia pneumoniae by a heterologous type III machinery

Chlamydia spp. are strictly intracellular pathogens that grow inside a vacuole, called an inclusion. They possess genes encoding proteins homologous to components of type III secretion machineries, which, in other bacterial pathogens, are involved in delivery of bacterial proteins within or through the membrane of eukaryotic host cells. Inc proteins are chlamydial proteins that are associated with the inclusion membrane and are characterized by the presence of a large hydrophobic domain in their amino acid sequence. To investigate whether Inc proteins and other proteins exhibiting a similar hydropathic profile might be secreted by a type III system, we used a heterologous secretion system. Chimeras were constructed by fusing the N-terminal part of these proteins with a reporter, the Cya protein of Bordetella pertussis, and these were expressed in various strains of Shigella flexneri. We demonstrate that these hybrid proteins are secreted by the type III secretion system of S. flexneri, thereby providing evidence that IncA, IncB and IncC are secreted by a type III mechanism in chlamydiae. Moreover, we show that three other proteins from Chlamydia pneumoniae, all of which have in common the presence of a large hydrophobic domain, are also secreted by S. flexneri type III secretion machinery.     

Import of bacterial pathogenicity factors into mitochondria

Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane beta-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors.     

Haemonchus contortus: evaluation of two signal sequence trapping systems for detection of secreted molecules

Given that signal sequences between secreted proteins of different species can be interchanged, it is reasonable to expect that both mammalian and yeast signal sequence trapping (SST) systems would secrete Haemonchus contortus proteins with similar efficiency and quality. To determine if H. contortus cDNAs that contain a signal sequence could re-establish secretion of a reporter protein, mammalian and yeast SST vectors were designed, 10 H. contortus genes selected, and their respective cDNAs cloned into these two SST vectors. The selected molecules included genes known to code for excretory/secretory or membrane-bound proteins as potential test 'positives', and genes known to code for non-secreted proteins as test 'negatives'. While differentiation between secretion and non-secretion was evident in both systems, the results indicated greater efficiency was achieved when the mammalian system was used. Therefore, mammalian SST using COS cells would be a more useful tool to screen H. contortus cDNA libraries for potential secreted and type-1 integral membrane proteins than yeast SST.     

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus.     

Prediction of protein signal sequences and their cleavage sites

Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The knowledge of signal sequences has become a crucial tool for pharmaceutical scientists who genetically modify bacteria, plants, and animals to produce effective drugs. However, to effectively use such a tool, the first important thing is to find a fast and effective method to identify the "zipcode" entity; this is also evoked by both the huge amount of unprocessed data available and the industrial need to find more effective vehicles for the production of proteins in recombinant systems. In view of this, a sequence-encoded algorithm was developed to identify the signal sequences and predict their cleavage sites. The rate of correct prediction for 1,939 secretory proteins and 1,440 nonsecretory proteins by self-consistency test is 90.14% and that by jackknife test is 90.13%. The encouraging results indicate that the signal sequences share some common features although they lack similarity in sequence, length, and even composition and that they are predictable to a considerably accurate extent.     

Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.     

The surprising complexity of signal sequences

Most secreted and many membrane proteins contain cleavable N-terminal signal sequences that mediate their targeting to and translocation across the endoplasmic reticulum or bacterial cytoplasmic membrane. Recent studies have identified many exceptions to the widely held view that signal sequences are simple, degenerate and interchangeable. Growing evidence indicates that signal sequences contain information that specifies the choice of targeting pathway, the efficiency of translocation, the timing of cleavage and even postcleavage functions. As a consequence, signal sequences can have important roles in modulating protein biogenesis. Based on a synthesis of studies in numerous experimental systems, we propose that substrate-specific sequence elements embedded in a conserved domain structure impart unique and physiologically important functionality to signal sequences.     

Membrane translocation of folded proteins

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.     

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.     

Secretion of proteins and assembly of bacterial surface organelles: shared pathways of extracellular protein targeting

Extracellular or surface localization of virulence determinants is an important attribute of pathogenic microorganisms. The past decade has seen significant research advances in defining the steps and identifying the necessary machinery for protein secretion from bacterial cells. In Gram-negative pathogens, four distinct classes of secretion pathways have been identified that deliver virulence factors to their sites of action. These pathways are responsible for the delivery of soluble extracellular enzymes into the surrounding medium, or for specifically targeting proteins to the host cell. In several instances protein secretion pathways are similar to those involved in assembly of bacterial appendages. Combination of biochemical and genetic analyses has recently revealed that the pathways of protein secretion and surface localization of various organelles are mechanistically similar which was not apparent simply by comparing amino acid sequences of related proteins. The choice of the pathway that a protein will utilize may not be dictated only by the specific requirement of the secreted protein to traverse the cell envelope in the functional form, but also by the need to assure its delivery to the correct site of action outside the bacterial cell.