Membrane-Bound Ost Subunits may Participate in the Disposal of Glycoproteins During Erad

The retrotranslocation of misfolded glycoproteins from the Endoplasmic Reticulum (ER) to the cytosol is a key step in the ER-associated degradation (ERAD) mechanism, which aims to prevent the accumulation of dysfunctional proteins in the early secretory pathway by targeting them for proteasomal degradation. The vast majority of newly-synthesized membranal and secretory proteins in mammalian cells are N-glycosylated at the site of translocation into the ER by the Oligosacharyl Transferase (OST) complex. The addition of a pre-assembled Glycan is the first post-translational modification event, executed in the ER, and is of great significance for the maturation and the quality control of these glycoproteins. Surprisingly, we found that during proteasomal inhibition and ERAD substrate accumulation, one of the OST subunits, the Defender Against Cell Death 1 (DAD1), relocalized to the ER Quality Control Compartment (ERQC), which has been proposed to be a site of retrotranslocation. Moreover, additional OST subunits were found to be part of the ERAD complex, formed during substrate accumulation, as seen by SILAC analysis. By pulse-chase experiments we found that the degradation of an ERAD substrate model, H2a accelerates in the presence of over-expressed DAD1 or of Robophorin1, an additional OST subunit. Consistently, by knocking down the non-essential TUSC3 subunit, the degradation of H2a was inhibited, while its N-glycosylation was not. Altogether our results suggest a possible role for the OST complex, known to be located at the forward-translocation site, during ERAD, perhaps in the retro-translocation event.