Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

Zhang, Sen; Finkelstein, Alan; Collier, R. John

doi:10.1073/pnas.0405754101

Cited by 119 publications

(159 citation statements)

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“…The fall in conductance at C20 mV upon addition of LF N to the cis solution is caused by the entrance of its N-terminal end into the (PA 63 ) 7 channel, thereby blocking it (Zhang et al 2004a). The blockage of ion flow through the channel is essentially total.…”

Section: Introductionmentioning

confidence: 99%

Proton-coupled protein transport through the anthrax toxin channel

Finkelstein

2008

Phil. Trans. R. Soc. B

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Anthrax toxin consists of three proteins (approx. 90 kDa each): lethal factor (LF); oedema factor (OF); and protective antigen (PA). The former two are enzymes that act when they reach the cytosol of a targeted cell. To enter the cytosol, however, which they do after being endocytosed into an acidic vesicle compartment, they require the third component, PA. PA (or rather its proteolytically generated fragment PA 63 ) forms at low pH a heptameric b-barrel channel, (PA 63 ) 7 , through which LF and OF are transported-a phenomenon we have demonstrated in planar phospholipid bilayers. It might appear that (PA 63 ) 7 simply forms a large hole through which LF and OF diffuse. However, LF and OF are folded proteins, much too large to fit through the approximately 15 Å diameter (PA 63 ) 7 b-barrel. This paper discusses how the (PA 63 ) 7 channel both participates in the unfolding of LF and OF and functions in their translocation as a proton-protein symporter.

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Section: Introductionmentioning

confidence: 99%

Proton-coupled protein transport through the anthrax toxin channel

Finkelstein

2008

Phil. Trans. R. Soc. B

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“…Arora and Lepplea (36) demonstrated that the deletion of amino acids 1-40 of lethal factor results in a complete loss of toxicity for macrophages. Recently, Zhang et al (37) found that the deletion of the N-terminal 27-to 36-aa residues from lethal factor inhibited acid-triggered translocation to the cytosol of target cells. Because these regions encompass the MEME motif in lethal factor, it is tempting to speculate that these sequences may play a role in the translocation process.…”

Section: P]-labeled Adpr-ef-2 (B)mentioning

confidence: 99%

A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol

Ratts

Trujillo

Bharti

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by BLAST, CLUSTAL W, and MEME computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB 389IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102͞6TG cells that express the T1 peptide, Hut102͞6TG-T1. In contrast to the parental cell line, Hut102͞6TG-T1 cells are ca. 10 4 -fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102͞ 6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit ␤ to this region of the diphtheria toxin transmembrane domain.early endosomes ͉ translocation ͉ ADP-ribosyltransferase ͉ coatomer protein complex subunit ␤ T he intoxication of eukaryotic cells by diphtheria toxin follows an ordered series of interactions between the toxin and cellular factors that lead to inhibition of protein synthesis and cell death (1). Biochemical, genetic, and x-ray crystallographic analysis of the toxin has shown the protein to be composed of three distinct domains: an N-terminal catalytic (C) domain, a central transmembrane (T) domain, and the C-terminal receptor-binding domain (2-5). The intoxication process is initiated by the binding of the toxin to its cell surface receptor, a heparin binding epidermal growth factor-like precursor, and CD9 (6, 7). Once bound to its receptor, the toxin is internalized by receptormediated endocytosis into an early endosomal compartment (8). Upon acidification of the endosomal lumen by vesicular ATPase, the T domain undergoes a conformational change and spontaneously inserts into the vesicular membrane, forming an 18-to 22-Å pore or channel (9, 10). It is widely believed that the C domain, in a fully denatured form, is specifically thread through this channel and released into the cytosol. Once the C domain is refolded into an active conformation, it catalyzes the NAD ϩ -dependent ADP ribosylation of elongation factor 2 (EF-2), causing irreversible inhibition of protein synthesis and death of the cell by apoptosis (11,12). Although the endosomal membrane translocation of the C domain is understood in broad terms, the precise molecular mechanism(s) of this step in the intoxication process has remained largely unknown.Two hypotheses for C ...

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“…The small volume of the droplets, typically ≤200 nanoliters, permits capture of translocated cargo molecules at concentrations orders of magnitude higher than in experiments with traditional planar bilayers (2,5,6,8), where compartment size is typically approximately 1 mL. The size of the bilayer can be adjusted by positioning the droplets relative to one another with the micromanipulators, and the ratio of bilayer area to capture volume may thereby be maximized (12,13).…”

mentioning

confidence: 99%

“…For some toxins there is considerable evidence that these pores are functionally relevant to the transport of cognate catalytic moieties ("cargo" proteins) across cellular membranes (3). In anthrax toxin, for example, cargo molecules block current through the pores formed by the protective antigen (PA) moiety, and after an appropriate electrical potential or a proton gradient is applied, the current is restored to the pore's open-state level, suggesting translocation of the cargo (5)(6)(7)(8). A number of control experiments support this interpretation (5,(7)(8)(9).…”

mentioning

confidence: 99%

“…In anthrax toxin, for example, cargo molecules block current through the pores formed by the protective antigen (PA) moiety, and after an appropriate electrical potential or a proton gradient is applied, the current is restored to the pore's open-state level, suggesting translocation of the cargo (5)(6)(7)(8). A number of control experiments support this interpretation (5,(7)(8)(9). Detecting and assaying cargo protein in the trans chamber, opposite to that to which the protein was added, has proven challenging, however, mainly because the quantities of the cargo believed to be translocated are so small.…”

mentioning

confidence: 99%

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Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

Fischer

Holden

Pentelute

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

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Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

Cited by 119 publications

References 38 publications

Proton-coupled protein transport through the anthrax toxin channel

Proton-coupled protein transport through the anthrax toxin channel

A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol

Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

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