Abstract
Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared 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 refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance 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 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process.
Highlights
The unfolded A1 subunit of cholera toxin (CT) enters the host cytosol by passing through a pore in the endoplasmic reticulum (ER) membrane
Control experiments using Fourier Transform Infrared (FTIR) spectroscopy demonstrated that ATP and ATP␥S had no direct effect on the structure of CTA1
Because ATP hydrolysis by heat shock protein 90 (Hsp90) is required to refold CTA1, our collective observations support a model of toxin translocation in which Hsp90 couples CTA1 refolding with CTA1 extraction from the ER
Summary
The unfolded A1 subunit of cholera toxin (CT) enters the host cytosol by passing through a pore in the endoplasmic reticulum (ER) membrane. The subsequent discovery of CTA1 conformational instability indicated the toxin could not independently refold at physiological temperature and required a host factor(s) for extraction to the cytosol [13, 30]. CTA1 passes through one or more ER translocon pores (38 – 42) in an unfolded state and must achieve an active conformation in the cytosol to modify its G protein target in the lipid rafts of the plasma membrane [43, 44]. An interaction with ARF proteins will enhance the activity of folded CTA1 and is required for productive intoxication of cultured cells, but ARF alone cannot induce a gain-of-structure or gain-of-function in disordered CTA1 [47]. These studies have elucidated a new, Hsp90driven mechanism for toxin translocation that may apply to a broad range of ADP-ribosylating toxins
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