COP I and II dependent trafficking controls ER-associated degradation in mammalian cells

Abstract

Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an established ERAD substrate, asialoglycoprotein receptor H2a, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increased retrotranslocation and ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

“…ER pre-QC has been described as a general protective mechanism of the ER in the presence of ER stress, and it is presumably regulated by activation of the ER unfolded protein response (UPR). 11,18,41 This model suggests that all ER stressors should induce ER pre-QC to a similar extent. We used proximity labeling combined with immunoblotting to determine whether ER stress, independently of the mechanism by which it is activated, always mistargets FLAG TTR (a known pre-QC substrate 12,25 ) into the cytosol.…”