Proline residues are a key determinant for toxin entry into the host cytosol - AB toxins consist of a catalytic A subunit and a cell-binding B subunit. They are released into the extracellular space but attack targets within the host cytosol. Entry into the cytosol only occurs after toxin endocytosis from the plasma membrane of the target cell. Some AB toxins, including pertussis toxin (PT) and cholera toxin (CT), travel to the endoplasmic reticulum (ER) before toxin disassembly and A chain passage into the cytosol. We have shown the dissociated A chain spontaneously unfolds at 37°C, which triggers its export to the cytosol through the mechanism of ER-associated degradation (ERAD). Most ERAD substrates are extracted to the cytosol through the action of the AAA ATPase p97, but we have shown that the catalytic subunits of PT (PTS1) and CT (CTA1) instead require the cytosolic chaperone Hsp90 for passage into the cytosol. Hsp90 was not thought to recognize any specific feature of an unfolded protein, but we identified an RPP binding motif for Hsp90 at the N-terminus of PTS1 and CTA1. Recent studies have documented a functional role for Grp94, the ER-localized Hsp90 family member, in PTS1 translocation and PT intoxication. Preliminary evidence further suggests the tight association between Grp94 and PTS1 is disrupted by cyclophilin B (CypB), an ER-localized peptidyl-prolyl cis-trans isomerase (PPI). Our resulting model for ERAD-mediated toxin translocation proposes that PTS1 proline residues drive toxin-chaperone interactions on both sides of the ER membrane. This novel concept will be examined using either loss-of-function studies in cell culture or in vitro studies on toxin structure, toxin-chaperone binding, and toxin translocation from ER-derived microsomes. We predict CypB and its PPI activity will be required for the release of Grp94 from PTS1, PTS1 export to the cytosol, and PT intoxication. Both Grp94 and Hsp90 will be required for PTS1 translocation and PT intoxication. These two chaperones will recognize distinct proline-containing regions of PTS1, and loss of either binding site will trap PTS1 in the ER. Grp94 will prevent the thermal unfolding of PTS1 but will not refold the toxin at 37°C, whereas Hsp90 will refold disordered PTS1 in an ATP-dependent manner that is sufficient for toxin extraction to the cytosol. This work could establish the importance of proline residues for toxin translocation by documenting a role for CypB in PTS1 export, a functional link between CypB and Grp94 in the processing of PTS1, and how PTS1 translocation involves the distinct functions of Hsp90 family members at both sides of the ER membrane. Cyclophilin and Hsp90 family members can be found in a complex and frequently act on the same substrates, but these coordinated functions have not yet been documented for ERAD-mediated toxin translocation. Both protein families represent promising pharmacological targets for treating whooping cough through the inactivation of PT. A deeper understanding of PTS1-chaperone interactions could thus lead to more precise and effective therapeutics against the specific proteins required for PTS1 translocation.
