The Ins and Outs of Cholera Toxin Retro-translocation.

[摘要] Cholera toxin (CT), the virulence factor of Vibrio cholerae, is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the CTA1 subunit retro-translocates to the cytosol to induce toxicity. To accomplish this, the toxin hijacks ER-associated degradation (ERAD) machinery that ejects misfolded proteins to the cytosol for ubiquitin-dependent proteasomal destruction. This thesis aims to elucidate the molecular mechanisms that promote retro-translocation of the toxin in both the ER lumen and in the cytosol.Our previous in vitro analyses demonstrated that the ER oxidoreductase protein disulfide isomerase (PDI) acts as a redox-dependent chaperone to unfold CTA1 and presumably to initiate toxin retro-translocation. Reduced PDI binds and unfolds CTA1 and subsequent oxidation of PDI by Ero1α enables toxin release. We show in cells that down-regulation of Ero1α decreases retro-translocation of CTA1 by increasing reduced PDI and blocking efficient toxin release. Overexpression of Ero1α also attenuates CTA1 retro-translocation by increasing PDI oxidation and preventing it from engaging the toxin effectively. Interestingly, Ero1α down-regulation increases interaction between PDI and Derlin-1, an ER membrane component of the retro-translocation complex. These findings demonstrate that an appropriate Ero1α-PDI ratio is critical for regulating the binding-release cycle of CTA1 during retro-translocation and implicate the redox state of PDI in targeting to the retro-translocon.Upon reaching the cytosol misfolded proteins are poly-ubiquitinated, extracted from the ER membrane, and marked for proteasomal degradation. Because cholera toxin is neither ubiquitinated nor degraded it is uncertain how it is released into the cytosol. To elucidate this mechanism we developed an in vitro assay that permits release of CTA1 from intact ER membranes upon addition of cytosolic extract. The toxin must be delivered to the ER and processed normally into CTA1 for release. Furthermore, heat inactivation and energy depletion of the extract indicate that a proteinaceous, energy-dependent activity exists. However, fractionation of the cytosol shows that this activity does not co-fractionate with several primary candidates for the retro-translocation factor. Finally, expansion of the assay to include TCRα reveals that substrate extraction and release from the ER membrane are two distinct steps of retro-translocation.

[发布机构] University of Michigan

[效力级别] Retro-translocation

[关键词] Cholera Toxin;Retro-translocation;ERAD;CTA1;Molecular;Cellular and Developmental Biology;Science;Cellular and Molecular Biology