title: Co- and post-translocation roles for HSP90 in cholera Intoxication.
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.
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32826 and.
authors.forename: Helen
authors.initials: H
authors.lastname: Burress
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32826 and.
authors.forename: Michael
authors.initials: M
authors.lastname: Taylor
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32826 and.
authors.forename: Tuhina
authors.initials: T
authors.lastname: Banerjee
authors.affiliation: the Department of Physics, University of Central Florida, Orlando, Florida 32816.
authors.forename: Suren A
authors.initials: SA
authors.lastname: Tatulian
authors.affiliation: From the Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32826 and kteter@mail.ucf.edu.
authors.forename: Ken
authors.initials: K
authors.lastname: Teter
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002772:Cholera Toxin / Q000737:chemistry* / Q000633:toxicity; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport; D020349:Surface Plasmon Resonance
chemical_list: D000089982:ADP-Ribosylation Factor 6; D018841:HSP90 Heat-Shock Proteins; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; C000715348:ARF6 protein, human
keywords: Cholera Toxin; Fourier Transform IR (FTIR); Heat Shock Protein 90 (Hsp90); Host-pathogen Interaction; Protein Translocation; Surface Plasmon Resonance (SPR)