Derlin-1 and the E3 Ubiquitin Ligases Hrd1 and gp78 Facilitate Cholera Toxin Retro-translocation.

Abstract

The endoplasmic reticulum (ER) is the site of folding and assembly for membrane and secretory proteins. When proteins fail to reach their proper conformations, the ER quality control system, ER-associated degradation (ERAD), is responsible for maintaining cell homeostasis via the identification, modification and transport (retro-translocation) of misfolded proteins from the ER to the cytosol for proteasomal degradation. As a model ERAD substrate, Cholera toxin (CT), produced by Vibrio cholerae, relies on its receptor-binding B subunit (CTB) to enter the ER of intestinal epithelial cells, where its catalytic A1 subunit (CTA1) hijacks the ERAD pathway to induce toxicity. This thesis focuses on understanding the role of membrane proteins in the retro-translocation of CTA1.

We found that two core components of the retro-translocon, Derlin-1 and the E3 ubiquitin ligases Hrd1 and gp78, facilitate the retro-translocation of CTA1. The expression of dominant negative variants of Derlin-1, Hrd1 and gp78 inhibits the ER-to-cytosol transport of CTA1. Derlin-1, Hrd1 and gp78 interact with CTB and CTA, as well as with the ER oxido-reductase PDI, a lumenal protein known for its role in unfolding the toxin prior to retro-translocation. Furthermore, we reveal a previously unknown role for CTB in the targeting of the toxin to this retro-translocation machinery in the ER membrane. Collectively, these findings suggest a model by which CTB targets the holotoxin to the retro-translocation machinery where PDI unfolds the toxin for retro-translocation through the ER membrane. The function of Hrd1 and gp78 in this model is perplexing, as CTA1 is presumed to be a non-ubiquitinated substrate. However, our data indicate that the catalytic activity of Hrd1, gp78 and an E2 ubiquitin conjugating enzyme dedicated to ERAD are essential for CTA1 transport. Thus, in addition to identifying novel ERAD components involved in CTA1 retro-translocation and elucidating a mechanism by which CT is targeted to the ERAD machinery, we demonstrate that similar to other known ERAD substrates, an intact ubiquitination system is necessary for the ER-to-cytosol transport of CTA1. These data provide important insight into the general mechanism of ERAD and further our understanding of how ERAD substrates utilize the retro-translocation machinery.