Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

Abstract

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...

Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells

“…Moreover, HSP90 was recently shown to mediate dislocation of the cholera toxin A1 (CTA1) subunit from the ER into the cytosol. ER-to-cytosol export of CTA1 requires association of HSP90 with CTA1, a process blocked by GA treatment (39). Thus, the Hsp90-mediated translocation system might have developed as a common mechanism in a variety of cell types, ultimately leading to an assortment of seemingly unrelated effects such as transcriptional activation (36), intoxication (38), and cross-presentation (this study).…”

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

“…Cytosolic Hsp90 has been shown to participate in the posttranslational targeting of preproteins to the TOC-TIC and translocase of the outer membrane-translocase of the inner membrane (TOM-TIM) import machinery of chloroplasts and mitochondria, respectively (30,40). It was recently reported that cytosolic Hsp90 participates in translocation of antigen across the endosomal membrane into the cytosol in mammalian cells and also in dislocation of the cholera toxin A1 subunit from the ER into the cytosol (41,42). We provide unique evidence for the involvement of an organellar Hsp90 in protein translocation (Figs.…”

Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit

“…The medium was removed and replaced with serum-free medium containing various concentrations of CT. After 2 h, the cells were processed for determination of cAMP levels as described in Ref. 34. The basal levels of cAMP determined from unintoxicated cells were background-subtracted from the values obtained for toxin-treated cells; the maximal response from the parental control cells was arbitrarily set to 100%; and all other results were expressed as ratios of that 100% value.…”

“…For the toxicity assay, cells were exposed to ice-cold acidic ethanol at 3 h post-transfection and were processed for determination of cAMP levels as described in (34). An additional set of transfected cells were chased overnight to determine the relative levels of CTA1 expression via metabolic labeling and immunoprecipitation.…”

“…For the translocation assay, cells were incubated at 24 h post-transfection in methionine-free medium for 1 h before [ 35 S]methionine was added for another hour. Digitonin was then used to generate separate membrane and cytosolic fractions as previously described (34). Both cellular fractions were immunoprecipitated with an anti-CTA antibody.…”