Translocation of a functional protein by a voltage-dependent ion channel

Slatin, Stephen L.; Nardi, Angèle; Jakes, Karen S.; Baty, Daniel; Duché, Denis

doi:10.1073/pnas.022480199

Cited by 28 publications

(20 citation statements)

References 32 publications

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“…This model is supported by the SPR data, which showed that the most appropriate model for FL-Tir binding to vesicles is a two-state conformational change, indicating initial binding followed by a structural rearrangement. This model of interaction has been observed previously for other membrane-inserted proteins (20,35,36). Following this insertion, the C-terminal domain of Tir could then bind to the membrane adjacent to the transmembrane regions (via the second cTir-located binding site) in an orientation such that an appropriate surface is presented to the cytoplasm for interaction with host cell proteins, which include specific kinases and the actin nucleating machinery.…”

Section: Tryptophan Fluorescence and Spr Reveal The Regions Of The Prmentioning

confidence: 69%

“…However, Tir localization to the membrane in vivo does not require the IBD region (6, 10), and combined with the data in this study, it seems likely that the transmembrane helices only are sufficient to promote insertion, with the intervening sequence playing no role but to undergo translocation across the membrane. This mechanism of membrane insertion and translocation has parallels with colicins, in which the native translocated region can be replaced with peptide epitopes (35) or even functional proteins (36), which become translocated upon insertion. Whether any sequence within the IBD is important in the insertion mechanism of Tir is currently being addressed.…”

Section: Tryptophan Fluorescence and Spr Reveal The Regions Of The Prmentioning

confidence: 99%

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Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Race

Lakey

Banfield

2006

Journal of Biological Chemistry

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Insertion of the enteropathogenic Escherichia coli Tir protein into the plasma membrane of intestinal epithelial cells is a crucial event in infection because it provides a receptor for intimate bacterial adherence. This interaction with the bacterial outer membrane protein intimin is also essential in generating a number of signaling activities associated with virulence. Tir can be modified at various sites by phosphorylation and functionally interacts with multiple host proteins. To investigate the mechanism of membrane insertion and to establish a model system in which the multiple interactions/ functions of Tir can be uncoupled and independently characterized, we used intrinsic tryptophan fluorescence, surface plasmon resonance, and protease digestion assays to show that Tir can insert directly into phospholipid vesicles in a composition-dependent manner to generate the topology reported in vivo. This is the first time that Tir has been shown to insert into membranes in a simple model system in the absence of chemical modification or other factors. These data are consistent with the protein interacting with lipids through two sites. The major site is localized to the transmembrane/intimin-binding domain region and includes Trp 235 , which is shown to be an effective reporter of interaction. The minor site is located within the C-terminal domain. Together, these data support a model in which Tir is released into the cytoplasm by the type III translocon and then independently inserts into the plasma membrane from a cytoplasmic location. A thorough understanding of this mechanism will be crucial to understand the subtleties of enteropathogenic E. coli pathogenesis. Enteropathogenic Escherichia coli (EPEC)3 is a major cause of infantile diarrheal disease in developing countries (1, 2) and is closely related to enterohemorrhagic E. coli (O157), which is an emerging health concern throughout the world (3). EPEC pathogenesis is dependent on (a) an active virulence-associated type III secretion system (TTSS) that directly injects "effector" proteins into host cells and (b) the interaction of Tir (translocated intimin receptor; one such effector protein), inserted into the host cell membrane, with the bacterial outer membrane protein intimin (recently reviewed in Refs. 4 and 5). The intimate adhesion of bacteria to host cell membranes as mediated by the intiminTir interaction is essential for virulence (6 -8) and results in subversion of host cell signaling processes promoting colonization of the intestinal tract (reviewed in Ref. 9).Following delivery by the TTSS, Tir inserts into the host cell plasma membrane, adopting a distinct topology composed of a large extracellular loop (the intimin-binding domain (IBD), residues 255-364) and N-terminal (residues 1-233) and C-terminal (residues 385-550) domains, which remain on the cytosolic face of the membrane (7, 10). These domains are linked by two predicted transmembrane helices (residues 234 -254 and 365-384). In this topology, the extracellular domain is available to ...

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Section: Tryptophan Fluorescence and Spr Reveal The Regions Of The Prmentioning

confidence: 69%

Section: Tryptophan Fluorescence and Spr Reveal The Regions Of The Prmentioning

confidence: 99%

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Race

Lakey

Banfield

2006

Journal of Biological Chemistry

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“…Another example is from the study of the voltagedependent gating of the colicin A channel, which involves a substantial structural rearrangement resulting in the transfer of about 35% of the 200 residues in its pore-forming domain across the membrane (44). Intriguingly, such a structural rearrangement of colicin A is capable of moving exogenous proteins of up to 134 residues across lipid bilayers and presenting them in a functional form to the trans solution (45). A basic mechanism of membrane insertion and topology reversion thus may be widely used by apparently distinct systems in protein translocation.…”

Section: Discussionmentioning

confidence: 99%

Dual Topology of the Escherichia coli TatA Protein

Gouffi¹,

Gérard²,

Santini

et al. 2004

Journal of Biological Chemistry

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“…All showed channel activity although the channels varied in size and gating behaviour in each case (data not shown). In a recent paper on the channel-forming toxin colicin A, it was reported that the DNase domain of colicin E2 did not induce channels in bilayers 18 . However, the experimental conditions used in these experiments (where channel events of the order of seconds were recorded) precludes their observation.…”

Section: Characterization Of Colicin Dnase Channelsmentioning

confidence: 99%

“…Structural rearrangements are also a pre-requisite for channelformation by the ten helical bundle, pore-forming colicins where a molten globule has been inferred to be the active species 36,37 . In the case of colicin Ia, it has been shown that the domain translocates large segments of polypeptide and even proteins across the bilayer during cytotoxic pore-formation 18,19,38 . While the channel activity of the E9 DNase is associated with the ability of colicin E9 to kill bacterial cells it cannot be the causative agent of cell death.…”

Section: Membrane Translocation By Dnase Colicinsmentioning

confidence: 99%

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

et al. 2002

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Article:Mosbahi, Khédidja, Lemaître, Christelle, Mobasheri, Hamid et al. (6 more authors) (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology. pp. 476-484. ISSN 1545-9985 https://doi.org/10.1038/nsb797 eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. White Rose Consortium ePrints Repositoryhttp://eprints.whiterose.ac.uk/ This is an author produced version of a paper published in Nature Structural Biology. This paper has been peer-reviewed but does not include final publisher proof-corrections or journal pagination.White Rose Repository URL for this paper: http://eprints.whiterose.ac.uk/archive/00001066/ Citation for the published paper Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nature Structural Biology, 9 (6). pp. 476-484. Citation for this paperTo refer to the repository paper, the following format may be used: Mosbahi, Khédidja and Lemaître, Christelle and Keeble, Anthony H. and Mobasheri, Hamid and Morel, Bertrand and James, Richard and Moore, Geoffrey R. and Lea, Edward J. A. and Kleanthous, Colin (2002) Colicin E9 is a 60 kDa toxin that is normally released from colicinogenic bacteria in the form of a heterodimeric complex with its 9.5 kDa immunity protein, Im9 (ref. 14). The immunity protein protects the colicin-producing bacterium from the activity of its own toxin but is jettisoned on entry of the colicin into a susceptible cell 15 . Hence, the form of the toxin tested in the bilayer experiments had the immunity protein removed (see Materials andMethods section). Previous work from our laboratory has shown that this form of the toxin retains complete biological activity 14 . Immunity-free colicin E9 (2 nM) was added to the cis chamber of a bilayer apparatus in 10 mM Tris/HCl buffer at pH 7.5, containing 0.1 M NaCl and 10 mM CaCl 2 , and a potential difference (p.d.) applied across the membrane. Random, fluctuating current was observed that showed evidence of opening and closing events with conductance of the order of ~100 pS, although larger conductance states were also seen ( Fig. 1a). In order to identify the region(s) of the protein responsible for this a...

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Translocation of a functional protein by a voltage-dependent ion channel

Cited by 28 publications

References 32 publications

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Insertion of the Enteropathogenic Escherichia coli Tir Virulence Protein into Membranes in Vitro

Dual Topology of the Escherichia coli TatA Protein

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

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