SummaryCholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by vesicular transport. In the ER, the catalytic CTA1 subunit dissociates from the holotoxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). It is hypothesized that CTA1 triggers its ERAD-mediated translocation into the cytosol by masquerading as a misfolded protein, but the process by which CTA1 activates the ERAD system remains unknown. Here, we directly assess the thermal stability of the isolated CTA1 polypeptide by biophysical and biochemical methods and correlate its temperature-dependent conformational state with susceptibility to degradation by the 20S proteasome. Measurements with circular dichroism and fluorescence spectroscopy demonstrated that CTA1 is a thermally unstable protein with a disordered tertiary structure and a disturbed secondary structure at 37°C. A protease sensitivity assay likewise detected the temperature-induced loss of native CTA1 structure. This protease-sensitive conformation was not apparent when CTA1 remained covalently associated with the CTA2 subunit. Thermal instability in the dissociated CTA1 polypeptide could thus allow it to appear as a misfolded protein for ERADmediated export to the cytosol. In vitro, the disturbed conformation of CTA1 at 37°C rendered it susceptible to ubiquitin-independent degradation by the core 20S proteasome. In vivo, CTA1 was also susceptible to degradation by a ubiquitin-independent proteasomal mechanism. ADPribosylation factor 6, a cytosolic eukaryotic protein that enhances the enzymatic activity of CTA1, stabilized the heat-labile conformation of CTA1 and protected it from in vitro degradation by the 20S proteasome. Thermal instability in the reduced CTA1 polypeptide has not been reported before, yet both the translocation and degradation of CTA1 may depend upon this physical property.

Na ؉ /H؉ exchangers (NHEs) mediate electroneutral exchange of Na ؉ for H ؉ and thereby play a central role in pH regulation and Na ؉ homeostasis. NHE3, the predominant epithelial isoform, is found in apical membranes of renal and intestinal epithelial cells, where it contributes to NaCl (re)absorption. NHE activity has been detected in endomembrane vesicles of epithelial cells, but the precise compartment involved and its functional role have not been defined. Many aspects of the targeting machinery that defines the compartmentation and polarity of epithelia are also functional in nonepithelial cells. We therefore compared the targeting of NHE1, the basolateral isoform, with that of NHE3 in Chinese hamster ovary cells. To circumvent the confounding effects of endogenous exchangers, epitope-tagged constructs of NHE1 and NHE3 were stably expressed in antiport-deficient (AP-1) cells. While NHE1 was found almost exclusively in the surface membrane, NHE3 was also found intracellularly, accumulating in a juxtanuclear compartment. Confocal microscopy showed this compartment to be distinct from the Golgi, trans-Golgi network, and lysosomes. Instead, NHE3 colocalized with transferrin receptors and with cellubrevin, markers of recycling endosomes. The activity of NHE3 in endomembranes was assessed by targeting pH-sensitive probes to the recycling endosomes using a chimeric cellubrevin construct with an accessible extracellular epitope. Fluorescence ratio imaging indicated that cellubrevin resides intracellularly in an acidic compartment. In AP-1 cells, endosomal acidification was unaffected by omission of Na ؉ but was dissipated entirely by concanamycin, a blocker of H ؉ -ATPases. In contrast, the cellubrevin compartment was more acidic in NHE3 transfectants, and the acidification was only partially reduced by concanamycin. Moreover, removal of extracellular Na ؉ resulted in a significant alkalization of the endocytic compartment. These results indicate that NHE3 is present and active in recycling endosomes. By recruiting NHE3 to the plasma membrane, modulation of vesicular traffic could contribute to the regulation of Na ؉ reabsorption across epithelia.

Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin. Cholera toxin (CT)2 is an AB 5 protein toxin that consists of a catalytic A moiety and a cell-binding B moiety (1, 2). The B subunit is pentameric ring-like structure that adheres to GM1 gangliosides on the plasma membrane of a target cell. The A subunit is initially synthesized as a 26 kDa protein that undergoes proteolytic nicking to generate a disulfide-linked A1/A2 heterodimer. The 21 kDa CTA1 polypeptide is an ADP-ribosyltransferase that modifies and activates Gs␣ in the host cell cytosol. CTA1 can be divided into three subdomains: the A1 1 subdomain contains the catalytic core of the toxin; the A1 2 subdomain is a short extended linker that connects the A1 1 and A1 3 subdomains; and the A1 3 subdomain is a globular structure with many hydrophobic residues as well as a cysteine residue involved with the single disulfide bridge between CTA1 and CTA2 (3). The 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the central pore of the B pentamer and thereby anchors CTA1 to CTB 5 . A ribbon diagram of the CT holotoxin which highlights the subdomain structure of CTA1 is provided in supplemental Fig. S1.To reach its cytosolic Gs␣ target, CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular traffic (4). A C-terminal KDEL sequence in the CTA2 subunit is thought to target and/or retain CT in the ER (4, 5). Conditions in the ER lead to reductive cleavage of the CTA1/CTA2 disulfide bond and chaperone-assisted dissociation of CTA1 from CTA2/CTB 5 (6 -9). Unfolding of the free A1 subunit then activates the quality control system of ER-associated degradation (ERAD), thereby promoting CTA1 translocation to the cytosol (10, 11). Most exported ERAD substrates are degraded by the ubiquitin-proteasome system, but it was hypothesized that CTA1 and the A chains of other ER-translocating toxins avoid t...

SummaryThe cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methylb b b b -cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N-linked glycosylation. Further translocation from the ER did not require the ER-associated degradation (ERAD) pathway, and was Derlin-1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double-stranded breaks was prevented in Brefeldin Atreated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.

Summary Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit - specifically, the loss of CTA1 tertiary structure at 37°C - triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel anti-toxin therapeutic agents.

Cholera toxin (CT) is an AB 5 toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1. Cholera toxin (CT)4 is one of the main virulence factors produced by Vibrio cholerae (1, 2). It is an AB-type protein toxin that contains separate catalytic and cell-binding subunits. The catalytic A subunit is initially synthesized as a 27 kDa protein, which undergoes proteolytic nicking to generate a disulfidelinked CTA1/CTA2 heterodimer. The ADP-ribosyltransferase activity of CT resides in the 22 kDa CTA1 polypeptide, while the 5 kDa CTA2 polypeptide maintains numerous non-covalent interactions with the B subunit and thereby links the enzymatic A1 moiety to the cell-binding B moiety. The CTB subunit, built from 11 kDa monomers, is a homopentameric ring-like structure that binds to GM1 gangliosides on the plasma membrane of a target cell.CT travels as an intact holotoxin from the cell surface to the ER (3). Environmental conditions in the ER facilitate reduction of the CTA1/CTA2 disulfide bond and dissociation of CTA1 from CTA2/CTB 5 . This process occurs at the resident redox state of the ER and involves the action of protein-disulfide isomerase (PDI), an ER-localized oxidoreductase (4 -8). Unfolding of the dissociated CTA1 subunit allows it to move into the cytosol through one or more protein-conducting channels in the ER membrane (9 -11). Cytosolic CTA1 then refolds into an active conformation and modifies its Gs␣ target.ER-associated degradation (ERAD), a host quality control mechanism, is responsible for the ER-to-cytosol dislocation of CTA1 (12)(13)(14). A variety of ER-localized chaperones, lectins, and oxidoreductases function in ERAD (15-17). These proteins recognize features that are present in misfolded proteins such as surface-exposed hydrophobic residues or improper patterns of N-linked glycosylation. When a misfolded protein is identified by the ERAD system, it is exported to the cytosol through Sec61 and/or Derlin-1 protein-conducting channels. Dislocated ERAD substrates are usually appended with polyubiquitin chains that serve as a molecular tag for degradation by the 26 S...

Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1-and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1-and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Verod2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before A chain passage into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP-ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.Pertussis toxin (PT) is an AB-type protein toxin that consists of an enzymatic A moiety and a cell-binding B moiety (reviewed in (1,2)). PT A (the S1 subunit) activates certain Gα proteins by an ADP-ribosylation reaction that utilizes NAD as a donor molecule. PT B is composed of an S2 subunit, an S3 subunit, two S4 subunits, and an S5 subunit. The oligomeric PT B complex forms a ring-like structure that is stable for temperatures up to 60ºC-70ºC (3). Non-covalent interactions position the catalytic S1 subunit within and on top of the B ring to form the PT holotoxin.PT B binds to glycoproteins or glycolipids on the plasma membrane of a target cell (4-6). The surface-bound toxin then travels by vesicular transport to the Golgi apparatus and most likely to the endoplasmic reticulum (ER) as well (7-12).