To cause disease, cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) lumen where the catalytic CTA1 subunit retro-translocates to the cytosol to induce pathological water secretion. Two retro-translocon components are the Derlins and ER-associated multi-spanning E3 ubiquitin ligases including Hrd1 and gp78. We demonstrated previously that Derlin-1 facilitates CTA1 retro-translocation. However, as CTA1 is neither ubiquitinated on lysines nor at its N-terminus, the role of E3 ligases in toxin retro-translocation is unclear. Here, we show that expression of mutant Hrd1 and gp78 and a mutant E2-conjugating enzyme dedicated to retro-translocation (Ube2g2) decrease CTA1 retro-translocation. Hrd1 knockdown also attenuated toxin retro-translocation. Binding studies demonstrate that Hrd1 and gp78 interact with CT and protein disulfide isomerase, an ER chaperone that unfolds CTA1 to initiate translocation. Moreover, we find that the toxin's association with Hrd1 and gp78 is blocked by dominant-negative Derlin-1, suggesting that CT is targeted initially to Derlin-1 and then transferred to Hrd1 and gp78. These data demonstrate a role of the E3 ubiquitin ligases in CTA1 retro-translocation, implicate a sequence of events experienced by the toxin on the ER membrane, and raise the possibility that ubiquitination is involved in the transport process.

Cholera toxin (CT) intoxicates cells by trafficking from the cell surface to the endoplasmic reticulum (ER), where the catalytic CTA1 subunit hijacks components of the ER-associated degradation (ERAD) machinery to retrotranslocate to the cytosol and induce toxicity. In the ER, CT targets to the ERAD machinery composed of the E3 ubiquitin ligase Hrd1-Sel1L complex, in part via the activity of the Sel1L-binding partner ERdj5. This J protein stimulates BiP's ATPase activity, allowing BiP to capture the toxin. Presumably, toxin release from BiP must occur before retrotranslocation. Here, using loss-and gain-of-function approaches coupled with binding studies, we demonstrate that the ER-resident nucleotide exchange factors (NEFs) Grp170 and Sil1 induce CT release from BiP in order to promote toxin retrotranslocation. In addition, we find that after NEF-dependent release from BiP, the toxin is transferred to protein disulfide isomerase; this ER redox chaperone is known to unfold CTA1, which allows the toxin to cross the Hrd1-Sel1L complex. Our data thus identify two NEFs that trigger toxin release from BiP to enable successful retrotranslocation and clarify the fate of the toxin after it disengages from BiP.