ER-to-cytosol polypeptide transport occurs when polypeptides are targeted for ER-associated degradation (ERAD). The necessity of ERAD is underscored by its importance in maintaining cellular homeostasis, but can also be induced due to aberrant ER quality control mechanisms, or to pathogen subversion of the host cellular machinery. ER-to-cytosol polypeptide transport can be delineated as four distinct processes: (1) recognition of ERAD substrates, (2) recruitment of cellular factors such as lectins, E2 ubiquitin conjugating enzymes, E3 ubiquitin ligases, and E4 chain-extension enzymes, (3) dislocation of the polypeptide through the dislocation pore, and (4) proteasome-mediated degradation. Many questions concerning the dislocation process remain unanswered: the identity of the dislocation pore has yet to be definitively identified, and cellular factors required for dislocation efficiency have not been fully characterized. To this end, we have sought to tease out factors that support dislocation. Our data demonstrate that translocating chain-associated membrane protein 1 (TRAM1) facilitates dislocation of membrane-integrated, but not soluble, ERAD substrates. TRAM1 allows lateral integration of nascent polypeptides into the ER membrane bilayer; similarly, TRAM1 probably allows lipid partitioning of polypeptides targeted for dislocation. In doing so, TRAM1 alleviates ER stress by preventing accumulation of misfolded proteins within the ER.

We also examined factors from the cytosolic face of the ER membrane. Ubiquitination is important in the process of dislocation, but the nature of ubiquitin conjugation and deconjugation is not fully understood. We utilized the dislocation inhibitor EERI to visualize novel pre-dislocation ERAD species, and postulate that a finite number of ubiquitin moieties, a characteristic that is substrate-dependent, acts as a signal for polypeptide extraction. On the other hand, we were also particularly interested in the effect of deubiquitinating enzymes (DUBs) on the process of dislocation. There are an estimated 100 DUBs in the human genome, but only a handful are involved in dislocation. We surmised that binding partners probably regulate activity of the DUBs examined. Therefore, we utilized the OTU domain of Crimean Congo Hemorrhagic Fever Virus (CCHFV) to non-specifically deubiquitinate polypeptides and examined ensuing effects on ERAD polypeptide dislocation. Our experiments demonstrate that increased deubiquitinating activity promotes dislocation. We postulate that OTU edits the ubiquitin chain length and/or type to allow correct chain formation for dislocation.