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

Michael Taylor,Fernando Navarro-Garcia,Jazmin Huerta,Helen Burress,Shane Massey,Keith Ireton,Ken Teter

doi:10.1074/jbc.m110.148981

Michael Taylor, Fernando Navarro-Garcia + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.148981

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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.

Highlights

Cholera toxin (CT)4 is one of the main virulence factors produced by Vibrio cholerae [1, 2]
Surface Plasmon Resonance (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

Summary

Introduction

Cholera toxin (CT)4 is one of the main virulence factors produced by Vibrio cholerae [1, 2]. To detect CTA1 in the cytosol or medium of toxin-treated cells, the SPR sensor slide was coated with an anti-CTA1 antibody [24]. NECA-treated cells were resistant to ricin, another AB toxin that uses the ERAD system for A chain dislocation to the cytosol (Fig. 3B).

Results

Conclusion