A protein export pathway involving Escherichia coli porins

1. Download : Download high-res image (296KB)
2. Download : Download full-size image

Highlights? The porin OmpF is a required element for the secretion of YebF ? YebF interacts with OmpF and OmpC channels at their periplasmic face ? YebF exhibits a dynamic surface that is involved in the secretion process ? Proposed model of YebF secretion mediated by OmpF     

The Tat protein export pathway

The Tat (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.     

Locking TolC entrance helices to prevent protein translocation by the bacterial type I export apparatus

The periplasmic entrance of the TolC channel tunnel is sealed by close-packing of inner and outer coiled-coils, and it has been proposed that opening of the entrance is achieved by an iris-like realignment of the inner coiled-coils. This is supported by experimental disruption of the key links connecting them, which effects transition to the open state in TolC inserted into planar lipid bilayers. Here we provide in vivo evidence for this "twist to open" mechanism by constraining the coiled coils with disulphide bonds, either self-locking or bridged by a chemical cross-linker, and reconstituting the resulting TolC variants into the type I protein export system in Escherichia coli. Introducing an intermonomer disulphide bridge between Ala159 and Ser350 caused a fivefold reduction in export, and when the coiled coils were cross-linked at the entrance constriction, between Asp374 of adjacent monomers or between Asn156 and Ala375, TolC-dependent export was abolished. In vivo cross-linking showed that the locked non-exporting TolC variants were still recruited to assemble the type I export apparatus. The data show that untwisting the entrance helices is essential for the export function of TolC in E.coli, specifically to allow access and passage of substrates engaged at the inner membrane translocase.     

The twin-arginine translocation (Tat) protein export pathway

The twin-arginine translocation (Tat) protein export system is present in the cytoplasmic membranes of most bacteria and archaea and has the highly unusual property of transporting fully folded proteins. The system must therefore provide a transmembrane pathway that is large enough to allow the passage of structured macromolecular substrates of different sizes but that maintains the impermeability of the membrane to ions. In the Gram-negative bacterium Escherichia coli, this complex task can be achieved by using only three small membrane proteins: TatA, TatB and TatC. In this Review, we summarize recent advances in our understanding of how this remarkable machine operates.     

A PIP Gets the plasmodium protein export pathway going

Survival of blood stage malaria parasites requires extensive host cell remodeling, which is facilitated by secretion of parasite proteins via a dedicated protein export pathway. In a recent Cell paper, Bhattacharjee et?al., (2012) describe PI(3)P binding as one of the first steps in targeting parasite proteins to the host cell.     

A new pathway for protein export in Saccharomyces cerevisiae

Several physiologically important proteins lack a classical secretory signal sequence, yet they are secreted from cells. To investigate the secretion mechanism of such proteins, a representative mammalian protein that is exported by a nonclassical mechanism, galectin-1, has been expressed in yeast. Galectin-1 is exported across the yeast plasma membrane, and this export does not require the classical secretory pathway nor the yeast multidrug resistance-like protein Ste6p, the transporter for the peptide a factor. A screen for components of the export machinery has identified genes that are involved in nonclassical export. These findings demonstrate a new pathway for protein export that is distinct from the classical secretory pathway in yeast.     

Overlapping functions of components of a bacterial Sec-independent protein export pathway.

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.     

On the role of TolC in multidrug efflux: the function and assembly of AcrAB–TolC tolerate significant depletion of intracellular TolC protein

TolC channel provides a route for the expelled drugs and toxins to cross the outer membrane of Escherichia coli. The puzzling feature of TolC structure is that the periplasmic entrance of the channel is closed by dense packing of 12 α‐helices. Efflux pumps exemplified by AcrAB are proposed to drive the opening of TolC channel. How interactions with AcrAB promote the close‐to‐open transition in TolC remains unclear. In this study, we investigated in vivo the functional and physical interactions of AcrAB with the closed TolC and its conformer opened by mutations in the periplasmic entrance. We found that the two conformers of TolC are readily distinguishable in vivo by characteristic drug susceptibility, thiol modification and proteolytic profiles. However, these profiles of TolC variants respond neither to the in vivo stoichiometry of AcrAB:TolC nor to the presence of vancomycin, which is used often to assess the permeability of TolC channel. We further found that the activity and assembly of AcrAB–TolC tolerates significant changes in amounts of TolC and that only a small fraction of intracellular TolC is likely used to support efflux needs of E. coli. Our findings explain why TolC is not a good target for inhibition of multidrug efflux.     

Unusual chemokine receptor antagonism involving a mitogen-activated protein kinase pathway

Antagonism of chemokines on chemokine receptors constitutes a new regulatory principle in inflammation. Eotaxin (CCL11), an agonist for CCR3 and an attractant of eosinophils, basophils, and Th2 lymphocytes, was shown to act as an antagonist for CCR2, which is widely expressed on leukocytes and is essential for inflammatory responses. In this report we provide direct evidence for a novel mechanism how chemokine receptor function can be arrested by endogenous ligands. We show that binding of eotaxin to CCR2 stimulates the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of the mitogen-activated protein kinase kinase 1/2-ERK pathway is indispensable for eotaxin-mediated attenuation of CCR2 function, as inhibition of ERK phosphorylation abolishes the arresting effect. ERK is also activated by CCR2 agonists, e.g., monocyte chemoattractant protein-1 (CCL2). However, the involved pathways are different, although in either case coupling of CCR2 to pertussis toxin-sensitive heterotrimeric G proteins is necessary. The results are in agreement with the view that CCR2 could assume different activation states depending on the ligand it encounters. With respect to actin polymerization and calcium mobilization, the different activation states lead to agonistic and antagonistic responses. It is conceivable that the intracellular signal transduction pathway that is activated by eotaxin could cause an attenuation of proinflammatory responses mediated by CCR2.     

ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.     

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.     

Following the leader: bacterial protein export through the Sec pathway.

Significant strides have been made during the past 20 years in our understanding of protein secretion across the bacterial inner membrane. Specialized chaperones select secretory polypeptide chains and usher them to a membrane-embedded preprotein translocase. This unique molecular machine envelops the polymeric substrate and migrates along its length in defined, energy-dependent steps. Consequently, preproteins are gradually pumped into the periplasm where they acquire their native, folded conformation.     

Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli.

A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex. This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment. HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59). Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase. The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC. Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate. The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential. Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function. The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it. In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e. TolC recruitment was not triggered. Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex.     

The liganding of glycolipid transfer protein is controlled by glycolipid acyl structure

Glycosphingolipids (GSLs) play major roles in cellular growth and development. Mammalian glycolipid transfer proteins (GLTPs) are potential regulators of cell processes mediated by GSLs and display a unique architecture among lipid binding/transfer proteins. The GLTP fold represents a novel membrane targeting/interaction domain among peripheral proteins. Here we report crystal structures of human GLTP bound to GSLs of diverse acyl chain length, unsaturation, and sugar composition. Structural comparisons show a highly conserved anchoring of galactosyl- and lactosyl-amide headgroups by the GLTP recognition center. By contrast, acyl chain chemical structure and occupancy of the hydrophobic tunnel dictate partitioning between sphingosine-in and newly-observed sphingosine-out ligand-binding modes. The structural insights, combined with computed interaction propensity distributions, suggest a concerted sequence of events mediated by GLTP conformational changes during GSL transfer to and/or from membranes, as well as during GSL presentation and/or transfer to other proteins.     

Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.     

Protein export through the bacterial flagellar type III export pathway

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly–disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Novel 3-dimensional dendrimer platform for glycolipid microarray

Glycolipids are important biological molecules that modulate cellular recognitions and pathogen adhesions. In this paper, we report a sensitive glycolipid microarray for non-covalently immobilizing glycolipids on a microarray substrate and we perform a set of immunoassays to explore glycolipid-protein interactions. This substrate utilizes a three-dimensional hydrazide-functionalized dendrimer monolayer attached onto a microscopic glass surface, which possesses the characteristics to adsorb glycoliplids non-covalently and facilitates multivalent attributes on the substrate surface. In the proof-of-concept experiments, gangliosides such as GM1, FucGM1, GM3, GD1b, GT1b, and GQ1b, and a lipoarabinomannan were tested on the substrate and interrogated with toxins and antibodies. The resulting glycolipid microarrays exhibited hypersensitivity and specificity for detection of glycolipid-protein interactions. In particular, a robust and specific binding of a pentameric cholera toxin B subunit to the GM1 glycolipid spotted on the array has demonstrated its superiority in sensitivity and specificity. In addition, this glycolipid microarray substrate was used to detect lipoarabinomannan in buffer within a limit-of-detection of 125 ng/mL. Furthermore, Mycobacterium tuberculosis (Mtb) Lipoarabinomannan was tested in human urine specimens on this platform, which can effectively identify urine samples either infected or not infected with Mtb. The results of this work suggest the possibility of using this glycolipid microarray platform to fabricate glycoconjugate microarrays, which includes free glycans and glycolipids and potential application in detection of pathogen and toxin.