Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared (FTIR) spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance (SPR) experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. This activity is consistent with its role as a chaperone that refolds endogenous cytosolic proteins as part of a foldosome complex consisting of Hsp90, Hop, Hsp40, p23, and Hsc70. A role for Hsc70 in CT intoxication has not yet been established. Here, biophysical, biochemical, and cell-based assays demonstrate Hsp90 and Hsc70 play overlapping roles in the processing of CTA1. Using SPR we determined that Hsp90 and Hsc70 could bind independently to CTA1 at distinct locations with high affinity, even in the absence of the Hop linker. Studies using isotope-edited FTIR spectroscopy found that, like Hsp90, Hsc70 induces a gain-of-structure in unfolded CTA1. The interaction between CTA1 and Hsc70 is essential for intoxication, as an RNAi-induced loss of the Hsc70 protein generates a toxin-resistant phenotype. Further analysis using isotope-edited FTIR spectroscopy demonstrated that the addition of both Hsc70 and Hsp90 to unfolded CTA1 produced a gain-of-structure above that of the individual chaperones. Our data suggest that CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with extraction from the ER. The subsequent binding of Hsc70 further refolds CTA1 in a manner not previously observed in foldosome complex formation. The interaction of CTA1 with these chaperones is essential to intoxication and this work elucidates details of the intoxication process not previously known.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-5825 |
| Date | 01 January 2014 |
| Creators | Burress, Helen |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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