Structure-Function Analysis of XcpP, a Component Involved in General Secretory Pathway-Dependent Protein Secretion in Pseudomonas aeruginosa

The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P. aeruginosa secretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.     

Protein import into chloroplasts: new aspects of a well-known topic

Protein import into plant chloroplasts is a fascinating topic that is being investigated by many research groups. Since the majority of chloroplast proteins are synthesised as precursor proteins in the cytosol, they have to be posttranslationally imported into the organelle. For this purpose, most preproteins are synthesised with an N-terminal presequence, which is both necessary and sufficient for organelle recognition and translocation initiation. The import of preproteins is facilitated by two translocation machineries in the outer and inner envelope of chloroplasts, the Toc and Tic complexes, respectively. Translocation of precursor proteins across the envelope membrane has to be highly regulated to react to the metabolic requirements of the organelle. The aim of this review is to summarise the events that take place at the translocation machineries that are known so far. In addition, we focus in particular on alternative import pathways and the aspect of regulation of protein transport at the outer and inner envelope membrane.     

Toc64--a preprotein-receptor at the outer membrane with bipartide function

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.     

Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus

Pseudomonas aeruginosa is able to translocate proteins across both membranes of the cell envelope. Many of these proteins are transported via the type II secretion pathway and adopt their tertiary conformation in the periplasm, which implies the presence of a large transport channel in the outer membrane. The outer membrane protein, XcpQ, which is involved in transport of folded proteins across the outer membrane of P . aeruginosa , was purified as a highly stable homomultimer. Insertion and deletion mutagenesis of xcpQ revealed that the C-terminal part of XcpQ is sufficient for the formation of the multimer. However, linker insertions in the N-terminal part can disturb complex formation completely. Furthermore, complex formation is strictly correlated with lethality, caused by overexpression of xcpQ . Electron microscopic evaluation of the XcpQ multimers revealed large, ring-shaped structures with an apparent central cavity of 95 Å. Purified PilQ, a homologue of XcpQ involved in the biogenesis of type IV pili, formed similar structures. However, the apparent cavity formed by PilQ was somewhat smaller, 53 Å. The size of this cavity could allow for the transport of intact type IV pili.     

Without a little help from 'my' friends: direct insertion of proteins into chloroplast membranes?

The chloroplast membranes are highly regulated and biological active regions of the living plant cell, which carry numerous essential proteinaceous components. For example, in the thylakoid membrane the photosynthesis apparatus, one of the most life-relevant biological machineries, is located. How these membrane proteins are targeted to and inserted into their target membranes was one of the questions we aimed to understand in the last few years. Fifteen years ago little to nothing was known about the targeting and translocation of outer envelope proteins (G.W. Schmidt and L.M. Mishkind, Annu. Rev. Biochem. 55 (1986)). Although several protein assisted pathways for translocation of proteins across the membranes have been characterised, only recent results gave insight into how membrane proteins are inserted into the chloroplast membranes. Here we will focus on the mode of insertion of a class of proteins into the outer envelope and the thylakoid membranes, which share a unique feature: they insert apparently directly into the lipid bilayer, i.e. without the help of a proteinaceous translocation pore.     

Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins

A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria. The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase. The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.     

From nuclear genes to chloroplast localized proteins

There is broad evidence that an endosymbiotic uptake of a cyanobacterial-type organism was the point of origin for the evolution of chloroplasts. During organelle evolution extensive gene transfer from the symbiont to the host genome occurred, which raises the question of how these gene products, namely proteins, which are still functional in chloroplasts, find their way back ‘home’. Nuclear-encoded proteins enter plastids via a complex import machinery that requires the coordinate interplay of a variety of soluble and membrane-bound factors on the cytosolic site as well as on the stromal side of the chloroplast envelope membranes. We define that the process called ‘import of chloroplast precursor proteins’ begins with the release of the polypeptide from the ribosomes and binding to cytosolic factors, such as a guidance complex, which accompanies (chaperones) proteins to chloroplasts. The translocation across the envelope membranes engages distinct translocation machineries at the outer and the inner envelope membranes. Additionally subsequent sorting events to different subcompartments within the plastids are operated by a number of distinct pathways, all of which seem to involve multiple subunits, which are largely of bacterial (symbiotic) origin. The evolutionary history of proteins mediating the import of chloroplast constituents across the envelope membranes seems more diverse. Since cyanobacteria lack a protein import pathway, it is not surprising that only a few subunits of the chloroplast translocon seem to be of symbiotic origin while others seem to be eukaryotic additions.     

Protein translocation into and across the chloroplastic envelope membranes

Post-translational protein import into chloroplasts follows a common route characterised by the need for nucleoside-triphosphates at various steps and two distinct protein import machineries at the outer and inner envelope membrane, respectively. Several subunits of these complexes have been elucidated. In contrast, protein translocation into the chloroplastic outer envelope uses distinct and various but poorly characterised insertion pathways. A topological framework for single-membrane spanning proteins of the chloroplastic outer envelope is presented.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

The xcp genes are required for protein secretion by Pseudomonas aeruginosa. They are involved in the second step of the process, i.e. the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion. The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd. Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo. Subcloning and complementation experiments showed that both proteins are required for secretion. The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae. These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae.     

Plastid division coordination across a double-membraned structure

Chloroplasts still retain components of the bacterial cell division machinery and research over the past decade has led to an understanding of how these stromal division proteins assemble and function as a complex chloroplast division machinery. However, during evolution plant chloroplasts have acquired a number of cytosolic division proteins, indicating that unlike the cyanobacterial ancestors of plastids, chloroplast division in higher plants require a second division machinery located on the chloroplast outer envelope membrane. Here we review the current understanding of the stromal and cytosolic plastid division machineries and speculate how two protein machineries coordinate their activities across a double-membraned structure.     

A new chloroplast protein import intermediate reveals distinct translocation machineries in the two envelope membranes: energetics and mechanistic implications

Chloroplast protein import presents a complex membrane traversal problem: precursor proteins must cross two envelope membranes to reach the stromal compartment. This work characterizes a new chloroplast protein import intermediate which has completely traversed the outer envelope membrane but has not yet reached the stroma. The existence of this intermediate demonstrates that distinct protein transport machineries are present in both envelope membranes, and that they are able to operate independently of one another under certain conditions. Energetic characterization of this pathway led to the identification of three independent energy-requiring steps: binding of the precursor to the outer envelope membrane, outer membrane transport, and inner membrane transport. Localization of the sites of energy utilization for each of these steps, as well as their respective nucleotide specificities, suggest that three different ATPases mediate chloroplast envelope transport.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex

N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.     

Protein secretion in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to have disseminated widely among Gram-negative bacteria.     

Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa.

Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa. Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features. Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane. To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization. We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.     

Dicarboxylic acid transport in Escherichia coli K12: involvement of a binding protein in the translocation of dicarboxylic acids across the outer membrane of the cell envelope.

We have previously found that the dicarboxylate transport system in Escherichia coli K12 is an active transport system and that at least one binding protein and two cytoplasmic membrane transport components are involved in the uptake of dicarboxylic acids. Recently, through surface labelling studies, some dicarboxylate binding proteins were found to be exposed on the cell surface. In the present paper, we demonstrate that the dicarboxylate transport component located in the outer membrane can be inactivated by two different kinds of nonpenetrating inhibitors, viz. proteases, and diazosulfanilic acid. These inhibitors seem to act on the dicarboxylate binding protein. By adding this protein to inactivated cells or to transport-negative mutants, we have succeeded in reconstituting the dicarboxylate transport system. These findings suggest that the dicarboxylate binding protein found on the cell surface plays an essential role in the translocation of dicarboxylic acids across the outer membrane.     

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

Background  

Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems.     

Protein transport machineries for precursor translocation across the inner mitochondrial membrane

The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.