Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Eriksen, Stig; Olsnes, Sjur; Sandvig, Kirsten; Sand, Olav

doi:10.1002/j.1460-2075.1994.tb06765.x

Cited by 31 publications

(33 citation statements)

References 42 publications

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“…The basic protocol for measuring current in these experiments represents a sensitive assay for pore formation in plasma membranes by PA and could probably be adapted for other pore-forming bacterial toxins (33,34). This whole-cell technique may ultimately prove suitable for the study of catalytic factor translocation.…”

Section: Discussionmentioning

confidence: 99%

Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current

Wolfe

Krantz

Rainey

et al. 2005

Journal of Biological Chemistry

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Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.Anthrax toxin, secreted by Bacillus anthracis, belongs to the A-B family of bacterial toxins and consists of three protein components. The "B" component, protective antigen (PA), 2 binds target cell receptors and is responsible for translocation of the two "A" components, lethal factor (LF) and edema factor (EF), to the cytoplasm of the affected cell (1). The action of LF and EF, a metalloprotease (2) and a soluble adenylyl cyclase (3), respectively, have various effects on cells, eventually leading to death of the infected host. PA is an 83-kDa protein that binds either of two known cellular receptors, capillary morphogenesis protein 2 (CMG2) (4) and tumor endothelial marker 8 (TEM8) (5). Cleavage of PA at a furin-sensitive site follows receptor binding and releases an N-terminal 20-kDa fragment, leaving a mature 63-kDa protein bound to the receptor (6). This allows interaction with other cleaved PA molecules and leads to the formation of ring-shaped, heptameric "prepore" structures on the cell surface (7). LF and EF bind competitively to the prepore, and the resulting complexes are internalized by endocytosis (8, 9). Endosome acidification by a vesicular membrane proton pump induces a conformational change in the prepore that allows it to form a membrane-spanning pore (10). LF and EF are believed to translocate through this pore to the cytosol. Because LF and EF are nontoxic in the absence of PA, a better understanding of the translocation process may reveal useful drug targets for disrupting the damaging effects of the toxin after anthrax infection.In vivo systems for the study of translocation in real time remain elusive, and instead, most relevant data on the process come from indirect biochemical studies (11) and from experiments in planar lipid bilayers (12, 13). The bio...

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Section: Discussionmentioning

confidence: 99%

Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current

Wolfe

Krantz

Rainey

et al. 2005

Journal of Biological Chemistry

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“…Studies in planar lipid bilayers have shown that under low pH conditions (pH Յ 6) in the cis compartment (the compartment to which protein is added), the isolated T domain is able to form cation-selective channels in the membrane (10); similar channels are formed by whole DT and by a truncated mutant, DT(CϩT), lacking the R domain (10,11). Channels are also formed in plasma membranes at low pH by the whole toxin or the B fragment (12,13). Two long hydrophobic helices (TH8 and TH9) buried within the native T domain insert into the bilayer when the domain converts to a transmembrane form (14)(15)(16)(17).…”

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confidence: 99%

Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain

Senzel

Collier

et al. 1999

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

130

141

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The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane, but how it does so, and whether cellular proteins are also required for this process, remain unknown. Here, we report results showing that the T domain alone is capable of translocating the entire C domain across model, planar phospholipid bilayers in the absence of other proteins. The T domain therefore contains the entire molecular machinery for mediating transfer of the catalytic domain of diphtheria toxin across membranes.Many toxic proteins of bacterial and plant origin are known to act by enzymically modifying substrates within the cytosol of mammalian cells, but the mechanism by which any of these toxins crosses a membrane to gain access to its substrates is not yet understood. These intracellularly acting toxins are generally bipartite proteins, containing the enzymic and receptorbinding functions on separate polypeptides, designated A and B, respectively (1). In some toxins, the B moiety also serves a second function, in mediating translocation of the A moiety across membranes.Diphtheria toxin (DT), the earliest example of an AB toxin, is a single, 535-residue polypeptide containing three folding domains: the amino-terminal C, or catalytic, domain (residues 1-185); the intermediate T, or transmembrane, domain (residues 202-378); and the carboxyl-terminal R, or receptorbinding, domain (residues 386-535) (Fig. 1). The catalytic domain is connected to the T domain by an arginine-rich loop and a readily reducible disulfide bridge (linking C186 to C201).After binding to its cell-surface receptor via the R domain, DT is proteolytically cleaved within the arginine-rich loop, yielding two disulfide-linked fragments: fragment A (corresponding to domain C) and fragment B (corresponding to domains T and R). The receptor-bound toxin is endocytosed and trafficked to an acidic vesicular compartment, where it undergoes a conformational change that allows the T domain to insert into the membrane and the catalytic domain to be translocated to the cytosol. The C186-C201 disulfide is reduced at some stage in this process, allowing the catalytic domain to be released into the cytosol. There, it catalyzes the ADP-ribosylation of elongation factor 2, causing inhibition of protein synthesis and cell death. (For a general review of diphtheria toxin, see ref. 2.)The crystallographic structure (3, 4) of native DT shows the T domain to consist of a bundle of 10 ␣-helices, which resembles the channel-forming domains of certain colicins (5-8) and ␣-helical domains found in certain mammalian proteins involved in regulation of apoptosis (9). Studies in planar lipid bilayers have shown that under low pH conditions (pH Յ 6) in the cis compartment (the compartment to which protein is added), the isolated T domain is able to form cation-selective channels in the membrane (10); similar channels are formed by whole DT and by a truncated mutant, DT(CϩT), lacking the R domain...

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“…By using whole cells exposed to low pH after DT binding at 4°C, mimicking the endosome environment (33,34), the toxin can be translocated directly from the cell surface to the cytosol when cells are incubated at 37°C. DT-induced pore formation has also been studied by measuring ion conductance or the release of radioactive ions from cells (35)(36)(37). By using these systems, the T domain of fragment B was shown to be important for insertion and translocation of fragment A.…”

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Diphtheria Toxin Translocation across Endosome Membranes

Umata

Mekada

1998

Journal of Biological Chemistry

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By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADPribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.Translocation of proteins across membranes is an important process in living cells. In eukaryotic cells proteins located in plasma membrane, lysosomes, and the Golgi complex are all synthesized on ribosomes attached to the endoplasmic reticulum, co-translationally translocated into the lumen of endoplasmic reticulum, and then transferred to appropriate destinations. Secreted proteins are also translocated into the endoplasmic reticulum, followed by exocytosis. Proteins localized in mitochondria, peroxisomes, chloroplasts, and nuclei are synthesized in the cytosol and translocated post-translationally into those organelles. The mechanisms of membrane penetration by these newly synthesized proteins and the machinery needed for their translocation have been studied in great detail.Translocation of proteins from outside the cell to inside also occurs. A number of protein toxins exert their toxic effects in mammalian cells by enzymatically modifying an intracellular target. At least a portion of the protein must be therefore be translocated across the cell membrane. Cholera toxin, Escherichia coli heat-labile toxin, and pertussis toxin ADP-ribosylate subunits of trimeric G proteins and cause constitutive activation of adenylate cyclases (1-3). Diphtheria toxin (DT) 1 and Pseudomonas aeruginosa exotoxin A inactivate elongation factor 2 and inhibit protein synthesis (4, 5). Several plant toxins as well as the Shiga toxin inactivate ribosomes by hydrolyzing specific sites in ribosome RNA (6 -8). Tetanus and botulinum neurotoxins cleave proteins that are part of the machinery for exocytosis in neurons (9). Other toxins, including Clostridium difficile toxin (10) and anthrax toxin (11), also affect intracellular targets.The mechanisms...

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Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Cited by 31 publications

References 42 publications

Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current

Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current

Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain

Diphtheria Toxin Translocation across Endosome Membranes

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