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• W2000655842 abstract "Cholera toxin (CT) is an AB5 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) is an AB5 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. IntroductionCholera toxin (CT) 4The abbreviations used are: CTcholera toxinBfAbrefeldin AERendoplasmic reticulumERADER-associated degradationGAgeldanamycinNECAN-ethylcarboxamidoadenosinePDIprotein-disulfide isomeraseSPRsurface plasmon resonance. is one of the main virulence factors produced by Vibrio cholerae (1De Haan L. Hirst T.R. Mol. Membr. Biol. 2004; 21: 77-92Crossref PubMed Scopus (173) Google Scholar, 2Sánchez J. Holmgren J. Cell Mol. Life Sci. 2008; 65: 1347-1360Crossref PubMed Scopus (167) Google Scholar). 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 disulfide-linked 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 (3Wernick N.L.B. Chinnapen D.J.-F. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar). Environmental conditions in the ER facilitate reduction of the CTA1/CTA2 disulfide bond and dissociation of CTA1 from CTA2/CTB5. This process occurs at the resident redox state of the ER and involves the action of protein-disulfide isomerase (PDI), an ER-localized oxidoreductase (4Lencer W.I. de Almeida J.B. Moe S. Stow J.L. Ausiello D.A. Madara J.L. J. Clin. Invest. 1993; 92: 2941-2951Crossref PubMed Scopus (81) Google Scholar, 5Majoul I. Ferrari D. Söling H.D. FEBS Lett. 1997; 401: 104-108Crossref PubMed Scopus (67) Google Scholar, 6Orlandi P.A. J. Biol. Chem. 1997; 272: 4591-4599Abstract Full Text Full Text PDF PubMed Google Scholar, 7Orlandi P.A. Curran P.K. Fishman P.H. J. Biol. Chem. 1993; 268: 12010-12016Abstract Full Text PDF PubMed Google Scholar, 8Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Unfolding of the dissociated CTA1 subunit allows it to move into the cytosol through one or more protein-conducting channels in the ER membrane (9Bernardi K.M. Forster M.L. Lencer W.I. Tsai B. Mol. Biol. Cell. 2008; 19: 877-884Crossref PubMed Scopus (85) Google Scholar, 10Schmitz A. Herrgen H. Winkeler A. Herzog V. J. Cell Biol. 2000; 148: 1203-1212Crossref PubMed Scopus (174) Google Scholar, 11Dixit G. Mikoryak C. Hayslett T. Bhat A. Draper R.K. Exp Biol Med. 2008; 233: 163-175Crossref PubMed Scopus (48) Google Scholar). Cytosolic CTA1 then refolds into an active conformation and modifies its Gsα target.ER-associated degradation (ERAD), a host quality control mechanism, is responsible for the ER-to-cytosol dislocation of CTA1 (12Teter K. Allyn R.L. Jobling M.G. Holmes R.K. Infect Immun. 2002; 70: 6166-6171Crossref PubMed Scopus (45) Google Scholar, 13Teter K. Holmes R.K. Infect Immun. 2002; 70: 6172-6179Crossref PubMed Scopus (69) Google Scholar, 14Teter K. Jobling M.G. Holmes R.K. Traffic. 2003; 4: 232-242Crossref PubMed Scopus (36) Google Scholar). A variety of ER-localized chaperones, lectins, and oxidoreductases function in ERAD (15Tsai B. Ye Y. Rapoport T.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 246-255Crossref PubMed Scopus (549) Google Scholar, 16Bar-Nun S. Curr. Top Microbiol. Immunol. 2005; 300: 95-125PubMed Google Scholar, 17Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (234) Google Scholar). 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 proteasome. However, CTA1 avoids the standard ERAD route of ubiquitin-dependent proteasomal degradation because it has a paucity of lysine residues for ubiquitin conjugation (18Hazes B. Read R.J. Biochemistry. 1997; 36: 11051-11054Crossref PubMed Scopus (277) Google Scholar, 19Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar).ERAD substrates are extracted from the ER through a mechanism that often involves the AAA ATPase Cdc48/p97 (16Bar-Nun S. Curr. Top Microbiol. Immunol. 2005; 300: 95-125PubMed Google Scholar). However, p97 appears to play a minimal role in CTA1 dislocation (20Kothe M. Ye Y. Wagner J.S. De Luca H.E. Kern E. Rapoport T.A. Lencer W.I. J. Biol. Chem. 2005; 280: 28127-28132Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 21McConnell E. Lass A. Wójcik C. Biochem. Biophys. Res. Commun. 2007; 355: 1087-1090Crossref PubMed Scopus (12) Google Scholar). An alternative model has proposed a ratchet mechanism, which involves the spontaneous refolding of CTA1 as it enters the cytosol (19Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar). Recent CTA1 structural studies do not support this model, as it has been shown that the isolated CTA1 subunit is a partially disordered, thermally unstable protein (22Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 23Ampapathi R.S. Creath A.L. Lou D.I. Craft Jr., J.W. Blanke S.R. Legge G.B. J. Mol. Biol. 2008; 377: 748-760Crossref PubMed Scopus (36) Google Scholar, 24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar). Because CTA1 is in an unfolded conformation at 37 °C, an as yet unidentified host protein must provide the driving force for CTA1 extraction from the ER.In this report we demonstrate that Hsp90 is required for CTA1 dislocation to the cytosol. The loss of Hsp90 function did not prevent CT transport to the ER but did inhibit the ER-to-cytosol export of CTA1. This, in turn, blocked CT activity against both cultured cells and ileal loops. Hsp90 has been shown to maintain membrane-embedded ERAD substrates in a soluble state and to help determine the fate of misfolded proteins (25Youker R.T. Walsh P. Beilharz T. Lithgow T. Brodsky J.L. Mol. Biol. Cell. 2004; 15: 4787-4797Crossref PubMed Scopus (127) Google Scholar, 26Wang X. Venable J. LaPointe P. Hutt D.M. Koulov A.V. Coppinger J. Gurkan C. Kellner W. Matteson J. Plutner H. Riordan J.R. Kelly J.W. Yates 3rd, J.R. Balch W.E. Cell. 2006; 127: 803-815Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). This work establishes a new role for Hsp90 in the extraction of a soluble ERAD substrate from the ER.RESULTS AND DISCUSSIONHsp90 functions in toxin translocation across the endosomal membrane (28Ratts R. Zeng H. Berg E.A. Blue C. McComb M.E. Costello C.E. vanderSpek J.C. Murphy J.R. J. Cell Biol. 2003; 160: 1139-1150Crossref PubMed Scopus (160) Google Scholar, 29Haug G. Aktories K. Barth H. Infect Immun. 2004; 72: 3066-3068Crossref PubMed Scopus (56) Google Scholar, 30Haug G. Leemhuis J. Tiemann D. Meyer D.K. Aktories K. Barth H. J. Biol. Chem. 2003; 278: 32266-32274Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), but its potential role in ERAD-mediated toxin dislocation has not been examined. To address this issue, SPR was used to determine whether Hsp90 could directly interact with the isolated CTA1 subunit at physiological temperature (Fig. 1A). Hsp90 binding to CTA1 occurred in an ATP-dependent manner that was blocked by GA. This demonstrated that Hsp90 could bind to CTA1 at 37 °C. Because CTA1 is in an unfolded conformation at 37 °C (22Pande A.H. Scaglione P. Taylor M. Nemec K.N. Tuthill S. Moe D. Holmes R.K. Tatulian S.A. Teter K. J. Mol. Biol. 2007; 374: 1114-1128Crossref PubMed Scopus (58) Google Scholar, 24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar), this also suggested that Hsp90 recognizes an unfolded conformation of CTA1 during the dislocation event. The interaction between CTA1 and Hsp90-ATP was not substantially affected by NECA, a drug that inhibits GRP94 but not Hsp90. GRP94, an ER-localized Hsp90, also bound to CTA1 in an ATP-dependent process that was blocked by both GA and NECA (Fig. 1B). The interaction between Hsp90 and CTA1 was much stronger than the interaction between GRP94 and CTA1: Hsp90 bound to CTA1 with a KD of 7 nm, whereas GRP94 bound to CTA1 with a KD of 292 nm (Fig. 2 and Table 1). Consistent with the dimeric natures of Hsp90 and GRP94 (31Zuehlke A. Johnson J.L. Biopolymers. 2010; 93: 211-217Crossref PubMed Scopus (175) Google Scholar), both chaperones bound to CTA1 in a 2:1 ratio of chaperone:toxin (Fig. 2). The specific, high-affinity interaction between Hsp90-ATP and CTA1 indicated that Hsp90 could be involved with the CT intoxication process.FIGURE 2Binding of CTA1 to Hsp90 or GRP94. A, Hsp90 was perfused over a CTA1-coated SPR sensor slide at 100, 400, 800, 1,600, and 3,200 nm concentrations. The ligand was removed from the perfusion buffer after 115 s. B, GRP94 was perfused over a CTA1-coated SPR sensor slide at 100, 200, 400, 800, and 1,600 nm concentrations. The ligand was removed from the perfusion buffer after 118 s. Note that the results for Hsp90 and GRP94 are plotted on different scales. For all conditions, ATP was present in the 37 °C perfusion buffer. Measurements collected from three independent experiments are shown. The orange lines represent best fit curves derived from the raw data using 1:2 (CTA1:Hsp90 or CTA1:GRP94) binding models. The SPR traces display, from top to bottom of the graph, results for decreasing concentrations of the ligand.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1CTA1 exhibits a high affinity interaction with Hsp90 and a lower affinity interaction with GRP94CTA1 binding partnerkakdKD1/Ms1/snmHsp909,9297.8 × 10−57GRP941,5374.5 × 10−4292 Open table in a new tab To detect a functional role for Hsp90 in CT intoxication, CT toxicity assays were performed in the presence or absence of GA (Fig. 3A). The elevated levels of cAMP resulting from CT intoxication were strongly inhibited in GA-treated cells. Whereas CT activity against untreated control cells exhibited a half-maximal effective concentration at 4 ng CT/ml, GA-treated cells only produced 36% of the maximal cAMP response when exposed to 100 ng CT/ml. Thus, at least 25-fold higher concentrations of CT would be required to elicit the same degree of intoxication in GA-treated cells as compared with the untreated control cells. In contrast, NECA-treated cells exhibited the same level of sensitivity to CT as the untreated control cells (Fig. 3A). GA-induced toxin resistance could not, therefore, be attributed to the inactivation of GRP94. NECA-treated cells were resistant to ricin, another AB toxin that uses the ERAD system for A chain dislocation to the cytosol (Fig. 3B). This observation was consistent with published results (32Spooner R.A. Hart P.J. Cook J.P. Pietroni P. Rogon C. Höhfeld J. Roberts L.M. Lord J.M. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17408-17413Crossref PubMed Scopus (62) Google Scholar) and demonstrated that NECA was functional at the concentration used in our CT assay. Additional control experiments demonstrated that GA did not block the cytopathic activity of a CTA1 construct that was expressed directly in the cytosol of cells transfected with a CTA1-encoding plasmid (27Teter K. Jobling M.G. Holmes R.K. Infect Immun. 2004; 72: 6826-6835Crossref PubMed Scopus (13) Google Scholar) (supplemental Fig. S1). The inhibitory effect of GA thus appeared to involve an event upstream of toxin-target interactions. Furthermore, GA did not inhibit the forskolin-induced elevation of intracellular cAMP: cells treated with GA and forskolin produced 100 ± 2% of the cAMP levels recorded for cells treated with forskolin alone (n = 3). Forskolin activates adenylate cyclase without the input of Gsα, so this observation demonstrated that GA did not directly inhibit the production of cAMP by adenylate cyclase.FIGURE 3GA but not NECA inhibits CT intoxication. A, CHO cells were incubated with varying concentrations of CT in the absence of additional treatment, in the presence of 0.1 μm GA, or in the presence of 0.1 μm NECA. After 2 h of continual toxin exposure, toxicity was assessed from the elevated levels of intracellular cAMP. The means ± S.E. of at least four independent experiments with triplicate samples are shown. B, CHO cells were incubated for 4 h with varying concentrations of ricin in the absence or presence of 0.1 μm NECA. Toxicity was then determined from the incorporation of [35S]methionine into newly synthesized proteins. The averages ± ranges of two independent experiments with triplicate samples are shown. C, surgically sealed sections of rat intestine were injected with 2 μg/ml of CT in the absence or presence of 15 μm GA. Two other loops were injected with only 15 μm GA or with PBS. Morphological examination (inset) and the calculation of fluid accumulation were performed 7 h post-injection. Results presented in the graph represent the averages ± S.D. of data obtained from 4 rats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)GA also inhibited CT activity in the ileal loop model of intoxication (Fig. 3C). Surgically sealed sections of intestine were injected with 2 μg/ml of CT in the absence or presence of GA. Seven hours later, the CT-injected loop displayed the distended morphology indicative of water accumulation resulting from productive intoxication. In contrast, loops that were injected with both CT and 15 μm GA exhibited substantially attenuated fluid accumulation and intestinal distension. GA derivatives have been evaluated as anti-cancer agents in clinical trials (33Powers M.V. Workman P. Endocr. Relat. Cancer. 2006; 13: S125-S135Crossref PubMed Scopus (255) Google Scholar, 34Blagosklonny M.V. Leukemia. 2002; 16: 455-462Crossref PubMed Scopus (229) Google Scholar). The protective effect of GA in a physiological model of intoxication suggests that it could also be used as a therapeutic to prevent or possibly treat cholera.Toxin resistance can result from an inhibition of toxin dislocation from the ER to the cytosol (9Bernardi K.M. Forster M.L. Lencer W.I. Tsai B. Mol. Biol. Cell. 2008; 19: 877-884Crossref PubMed Scopus (85) Google Scholar, 11Dixit G. Mikoryak C. Hayslett T. Bhat A. Draper R.K. Exp Biol Med. 2008; 233: 163-175Crossref PubMed Scopus (48) Google Scholar, 13Teter K. Holmes R.K. Infect Immun. 2002; 70: 6172-6179Crossref PubMed Scopus (69) Google Scholar, 24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar). To determine if GA blocked toxin export to the cytosol, we used an established dislocation assay (24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 35Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar) to monitor the appearance of CTA1 in the cytosol of intoxicated HeLa cells (Fig. 4). Like CHO cells, GA-treated HeLa cells were protected from CT as assessed by cAMP production (data not shown). After a 30 min exposure to CT at 4 °C, HeLa cells were chased for 2 h at 37 °C in the absence of additional toxin. Separate organelle and cytosol fractions were then collected from digitonin-permeabilized cells. Control experiments demonstrated the fidelity of our fractionation protocol: Western blot analysis detected the majority of Hsp90 in the supernatant (i.e. cytosolic) fraction, while PDI, a soluble ER protein, was found exclusively in the pellet fraction, which contained the intact ER and other membranes (Fig. 4A). After background subtraction, semi-quantitative analysis of the PDI Western blot detected a negligible amount of PDI (0.5%) in the supernatant fraction (n = 3). Additional Western blot analysis with non-reducing SDS-PAGE was performed in order to track the intracellular localization of CTA1 (Fig. 4B). The 21 kDa CTA1 subunit is initially synthesized as part of a larger, 26 kDa CTA precursor (1De Haan L. Hirst T.R. Mol. Membr. Biol. 2004; 21: 77-92Crossref PubMed Scopus (173) Google Scholar, 2Sánchez J. Holmgren J. Cell Mol. Life Sci. 2008; 65: 1347-1360Crossref PubMed Scopus (167) Google Scholar). Proteolytic nicking of CTA generates a disulfide-linked CTA1/CTA2 heterodimer, which is reductively cleaved in the ER; only the reduced CTA1 subunit enters the cytosol. Consistent with these observations, we detected both disulfide-linked CTA1/CTA2 and reduced CTA1 in the organelle fractions but only detected reduced CTA1 in the cytosol fractions. In comparison to the untreated control cells, the distribution of cytosolic CTA1 was unaffected by NECA treatment. However, GA-treated cells contained less cytosolic CTA1 than either untreated or NECA-treated cells. It thus appeared that Hsp90 function was required for efficient passage of CTA1 into the cytosol.FIGURE 4GA but not NECA inhibits CTA1 dislocation to the cytosol. HeLa cells were pulse-labeled at 4 °C for 30 min with 1 μg/ml of CT. The cells were then chased for 2 h at 37 °C in toxin-free medium containing no additions, 0.1 μm GA, 0.1 μm NECA, or 5 μg/ml of BfA. Permeabilization of the plasma membrane with digitonin was used to partition cell extracts into separate organelle (pellet; P) and cytosolic (supernatant; S) fractions. A, both fractions from untreated cells were probed by Western blot to establish the distributions of cytosolic marker Hsp90 and ER marker PDI. B, both fractions from untreated (no treatment) or drug-treated cells were probed for the presence of CTA1 by Western blot analysis of non-reducing SDS-PAGE gels. The upper band represents the disulfide-linked CTA1/CTA2 heterodimer; the middle band results from nonspecific cross-reactivity; and the lower band represents reduced CTA1. C, an SPR sensor slide coated with an anti-CTA antibody was used to detect the cytosolic pool of CTA1 from untreated or drug-treated cells. D, media samples taken from cells at the end of the chase were perfused over an SPR sensor slide coated with an anti-CTA antibody. For both C and D, CTA standards were perfused over the sensor slide as positive controls. The cytosolic fraction and medium from unintoxicated cells were also perfused over the sensor slides as negative controls for panels C and D, respectively. At the end of each experiment, bound sample was stripped from the sensor slide. One of four representative experiments is shown for each SPR experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To strengthen our Western blot analysis with an alternative and quantitative detection method, we employed the technique of SPR. HeLa cells exposed to CT at 4 °C were again fractionated into membrane and cytosolic components after a 2 h chase at 37 °C. In this experiment, the cytosolic fractions were perfused over a SPR sensor slide that had been coated with an anti-CTA antibody (Fig. 4C). A negligible background signal was obtained from unintoxicated cells, whereas control cells intoxicated in the absence of drug treatment produced a signal that was comparable to the response obtained from the 0.1-ng CTA standard (n = 4). The low level of cytosolic CTA1 in control cells was consistent with the known inefficiency of CT trafficking from the cell surface to the ER dislocation site (4Lencer W.I. de Almeida J.B. Moe S. Stow J.L. Ausiello D.A. Madara J.L. J. Clin. Invest. 1993; 92: 2941-2951Crossref PubMed Scopus (81) Google Scholar, 7Orlandi P.A. Curran P.K. Fishman P.H. J. Biol. Chem. 1993; 268: 12010-12016Abstract Full Text PDF PubMed Google Scholar, 36Fishman P.H. J. Cell Biol. 1982; 93: 860-865Crossref PubMed Scopus (36) Google Scholar). NECA-treated cells produced an SPR signal similar to the response obtained from the untreated control cells. In contrast, GA-treated cells produced an SPR signal that was substantially less than the response obtained from the untreated control cells. Exposure of untreated or GA-treated cells to proteasome inhibitors did not alter these results, which indicated the data were not affected by potential toxin degradation in the cytosol (supplemental Fig. S2). The detection of a minor pool of cytosolic CTA1 in GA-treated cells by SPR but not by Western blot reflects the greater level of sensitivity provided by SPR-based assays. CTA1 was not detected in the cytosol of cells treated BfA, a drug that prevents CT transport from the cell surface to the ER dislocation site (4Lencer W.I. de Almeida J.B. Moe S. Stow J.L. Ausiello D.A. Madara J.L. J. Clin. Invest. 1993; 92: 2941-2951Crossref PubMed Scopus (81) Google Scholar, 7Orlandi P.A. Curran P.K. Fishman P.H. J. Biol. Chem. 1993; 268: 12010-12016Abstract Full Text PDF PubMed Google Scholar, 37Nambiar M.P. Oda T. Chen C. Kuwazuru Y. Wu H.C. J. Cell. Physiol. 1993; 154: 222-228Crossref PubMed Scopus (65) Google Scholar).Protein concentration is directly proportional to the SPR-derived association rate constant (ka) (38Homola J. Anal. Bioanal. Chem. 2003; 377: 528-539Crossref PubMed Scopus (1809) Google Scholar), so we calculated the levels of cytosolic CTA1 from a plot of the ka values for the CTA standards. With this method, we estimated that 2.4-fold less CTA1 was in the cytosol of GA-treated cells than in the cytosol of untreated or NECA-treated cells (supplemental Table S1). A substantial inhibition of intoxication resulting from reduced, but not absent, levels of cytosolic CTA1 has previously been reported (13Teter K. Holmes R.K. Infect Immun. 2002; 70: 6172-6179Crossref PubMed Scopus (69) Google Scholar, 14Teter K. Jobling M.G. Holmes R.K. Traffic. 2003; 4: 232-242Crossref PubMed Scopus (36) Google Scholar, 24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar). The degree of intoxication appears to be influenced by numerous factors, including (i) the extent of CTA1 dislocation to the cytosol; (ii) the rate of CTA1 degradation in the cytosol; (iii) CTA1 activity against Gsα; and (iv) the de-activation of ADP-ribosylated Gsα by proteolysis or by the action of ADP-ribosyl(arginine)protein hydrolase (9Bernardi K.M. Forster M.L. Lencer W.I. Tsai B. Mol. Biol. Cell. 2008; 19: 877-884Crossref PubMed Scopus (85) Google Scholar, 13Teter K. Holmes R.K. Infect Immun. 2002; 70: 6172-6179Crossref PubMed Scopus (69) Google Scholar, 14Teter K. Jobling M.G. Holmes R.K. Traffic. 2003; 4: 232-242Crossref PubMed Scopus (36) Google Scholar, 19Rodighiero C. Tsai B. Rapoport T.A. Lencer W.I. EMBO Rep. 2002; 3: 1222-1227Crossref PubMed Scopus (115) Google Scholar, 24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 39Chang F.H. Bourne H.R. J. Biol. Chem. 1989; 264: 5352-5357Abstract Full Text PDF PubMed Google Scholar, 40Kato J. Zhu J. Liu C. Moss J. Mol. Cell. Biol. 2007; 27: 5534-5543Crossref PubMed Scopus (47) Google Scholar, 41Wernick N.L. De Luca H. Kam W.R. Lencer W.I. J. Biol. Chem. 2010; 285: 6145-6152Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The balance between these factors usually favors CTA1 activity against Gsα and, thus, productive intoxication. However, as shown here and in previous work, this balance can be shifted to protect cells from intoxication without completely eliminating the cytosolic pool of CTA1.To ensure that GA did not inhibit the intracellular trafficking of CT, we screened media samples from toxin-treated cells for the presence of free CTA1. The CT holotoxin travels by vesicle carriers from the cell surface to the ER (3Wernick N.L.B. Chinnapen D.J.-F. Cho J.A. Lencer W.I. Toxins. 2010; 2: 310-325Crossref PubMed Scopus (135) Google Scholar). Reduction and chaperone-assisted dissociation of CTA1 from the rest of the toxin then allows the free A1 subunit to move from the ER to the cytosol. In addition, a fraction of dissociated CTA1 enters the secretory pathway and is released into the extracellular medium (24Massey S. Banerjee T. Pande A.H. Taylor M. Tatulian S.A. Teter K. J. Mol. Biol. 2009; 393: 1083-1096Crossref PubMed Scopus (33) Google Scholar, 36Fishman P.H. J. Cell Biol. 1982; 93: 860-865Crossref PubMed Scopus (36) Google Scholar). Consistent with these observations, our SPR assay detected the secretion of CTA1 from intoxicated HeLa cells (Fig. 4D). Nearly equivalent amounts of CTA1 were secreted into the medium of untreated cells and GA-treated cells. No signal was obtained from the medium of BfA-treated cells, thus demonstrating that toxin trafficking to the ER was a prerequisite for CTA1 secretion into the medium. The aforementioned results were obtained with a SPR sensor slide that had been coated with an anti-CTA antibody. When the media samples were perfused over an SPR sensor slide that had been coated with an antibody against the cell-binding CTB subunit, no positive signals were obtained (data not shown). Thus, the secreted toxin was free CTA1 and not the CT holotoxin. Because untreated and GA-treated cells released equivalent amounts of free CTA1, GA did not appear to block holotoxin trafficking to the ER, CTA1 dissociation from the rest of the toxin in the ER, or the secretion of free CTA1.To further demonstrate that GA inhibited the CTA1 dislocation event rather than an upstream CT trafficking step, we combined our dislocation assay with a plasmid-based system to express CTA1 directly in the ER of transfected CHO cells (12Teter K. Allyn R.L. Jobling M.G. Holmes R.K. Infect Immun. 2002; 70: 6166-6171Crossref PubMed Scopus (45) Google Scholar, 27Teter K. Jobling M.G. Holmes R.K. Infect Immun. 2004; 72: 6826-6835Crossref PubMed Scopus (13) Google Scholar). With this system, CTA1 is co-translationally inserted into the ER and then dislocated back to the cytosol. Similar strategies have been used by other labs to monitor toxin dislocation (10Schmitz A. Herrgen H. Winkeler A. Herzog V. J. Cell Biol. 2000; 148: 1203-1212Crossref PubMed Scopus (174) Google Scholar, 35Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (99) Google Scholar, 42Castro M.G. McNamara U. Carbonetti N.H. Cell Microbiol. 2001; 3: 45-54Crossref PubMed Scopus (36) Google Scholar, 43LaPointe P. Wei X. Gariépy J. J. Biol. Chem. 2005; 280: 23310-23318Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 44Simpson J.C. Roberts L.M. Römisch K. Davey J. Wolf D.H. Lord J.M. FEBS Lett. 1999; 459: 80-84Crossref PubMed Scopus (130) Google S" @default.
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