Decoding the retrotranslocation step of ERAD in a mammalian system

Endoplasmic Reticulum associated degradation (ERAD) of terminally misfolded proteins plays an important role in maintaining ER homeostasis. Although being widely studied, several steps in the mechanism of this process remain unclear. One such step is the retrotranslocation of terminally misfolded proteins from ER to cytosol for its proteasome-dependent degradation. Many studies have tried to decipher the retrotranslocation mechanism using yeast as the model system. However, its exact mechanism, especially in the mammalian system, remains unknown. In this study I have used a novel Biotin-acceptor-peptide (BAP) – Biotin Ligase (BirA) approach and applied it together with an ERAD model substrate H2a (asialoglycoprotein receptor 2) to better understand the mechanism behind mammalian retrotranslocation. We hypothesise that upon retrotranslocation, the luminal tail of a membrane ERAD substrate will first be exposed to the cytosol via a retrotranslocation complex. Based on the above hypothesis, H2a (type II membrane protein) was tagged on its C-terminus with a BAP peptide, therefore upon retrotranslocation, H2a-BAP can be specifically biotinylated by an exogenously expressed BirA in the cytosol. I observed that interference with N-glycan processing, specifically mannose trimming inhibited targeting of the ERAD substrate, H2a-BAP, to the retrotranslocation complex. Next, I interfered with proteins which are predicted to be involved in the retrotranslocation, OS-9 (an ER lectin), Sel1L (adaptor protein of Hrd1), Hrd1 (E3 Ubiquitin Ligase), Derlin1 (rhomboid pseudoprotease), and Fbs2 (a cytosolic lectin component of SCFFbs2 ubiquitin ligase). My results lead to interesting insights in the mechanism and dynamics of retrotranslocation.