HSC70 and HSP90 chaperones perform complementary roles in translocation of the cholera toxin A1 subunit from the endoplasmic reticulum to the cytosol

Helen Burress,Alisha Kellner,Jessica Guyette,Suren A Tatulian,Ken Teter

doi:10.1074/jbc.ra119.008568

Helen Burress, Alisha Kellner + Show 3 more

Open Access

https://doi.org/10.1074/jbc.ra119.008568

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Abstract

Cholera toxin (CT) travels by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where the catalytic A1 subunit of CT (CTA1) dissociates from the rest of the toxin, unfolds, and moves through a membrane-spanning translocon pore to reach the cytosol. Heat shock protein 90 (HSP90) binds to the N-terminal region of CTA1 and facilitates its ER-to-cytosol export by refolding the toxin as it emerges at the cytosolic face of the ER membrane. HSP90 also refolds some endogenous cytosolic proteins as part of a foldosome complex containing heat shock cognate 71-kDa protein (HSC70) and the HSC70/HSP90-organizing protein (HOP) linker that anchors HSP90 to HSC70. We accordingly predicted that HSC70 and HOP also function in CTA1 translocation. Inactivation of HSC70 by drug treatment disrupted CTA1 translocation to the cytosol and generated a toxin-resistant phenotype. In contrast, the depletion of HOP did not disrupt CT activity against cultured cells. HSC70 and HSP90 could bind independently to disordered CTA1, even in the absence of HOP. This indicated HSP90 and HSC70 recognize distinct regions of CTA1, which was confirmed by the identification of a YYIYVI-binding motif for HSC70 that spans residues 83-88 of the 192-amino acid CTA1 polypeptide. Refolding of disordered CTA1 occurred in the presence of HSC70 alone, indicating that HSC70 and HSP90 can each independently refold CTA1. Our work suggests a novel translocation mechanism in which sequential interactions with HSP90 and HSC70 drive the N- to C-terminal extraction of CTA1 from the ER.

Highlights

Cholera toxin (CT) travels by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where the catalytic A1 subunit of CT (CTA1) dissociates from the rest of the toxin, unfolds, and moves through a membrane-spanning translocon pore to reach the cytosol
This novel, Heat shock protein 90 (HSP90)-driven translocation mechanism could apply to endogenous host proteins that move from the ER to the cytosol in a p97-independent process [19, 20] and to AB toxins such as diphtheria toxin (DT) that move from the endosomes to the cytosol [21,22,23,24,25,26]
The individual HSC70, Heat shock protein 70 (HSP70), and HSP90-organizing protein (HOP) proteins were dispensable for CT activity against cultured cells

Summary

To whom correspondence should be addressed

HSP90 only recognizes the disordered conformation of CTA1, and ATP hydrolysis by HSP90 is required for both CTA1 refolding and CTA1 translocation to the cytosol [18] This novel, HSP90-driven translocation mechanism could apply to endogenous host proteins that move from the ER to the cytosol in a p97-independent process [19, 20] and to AB toxins such as diphtheria toxin (DT) that move from the endosomes to the cytosol [21,22,23,24,25,26]. HSP90 can bind directly to CTA1 in an unusual interaction that does not require co-chaperones [17, 18], but we still predicted that HSC70 and HOP would be involved with the ER-to-cytosol export of CTA1. Our work demonstrates atypical client-chaperone interactions, independent of the foldosome complex, drive CTA1 export from the ER to the cytosol

Results

Discussion

15 Ϯ 2 32 Ϯ 1 17 Ϯ 1

Experimental procedures