Elucidation of the late steps in the glycan-dependent ERAD of soluble misfolded glycoproteins.

Abstract

The endoplasmic reticulum (ER) utilizes ER-associated degradation (ERAD), a highly conserved eukaryotic pathway, to eliminate misfolded or unassembled proteins and maintain protein homeostasis in cells. The clearance of misfolded glycoproteins involves several distinct steps, including the recognition of a specific glycan signal, retrotranslocation to the cytosol, and subsequent degradation of the misfolded protein by the ubiquitin proteasome system. Confocal microscopy was used to track the fate of a well-characterized ERAD substrate via a self-complementing split fluorescent protein assay. The results demonstrate that a misfolded variant of the STRUBBELIG (SUB) extracellular protein domain (SUBEX-C57Y) is retrotranslocated to the cytosol when transiently expressed in Nicotiana benthamiana leaf epidermal cells. Retrotranslocation requires a protein domain with a lesion that is exposed in the lumen of the ER, N-glycan trimming by α-mannosidases, HRD1-mediated ubiquitination, and the ATPase function of CDC48. The retrotranslocated SUBEX-C57Y ERAD substrate undergoes deglycosylation, and proteasomal degradation is blocked by a catalytically inactive cytosolic peptide N-glycanase. These findings define distinct aspects of ERAD that have been elusive until now and may represent the default pathway for degrading misfolded glycoproteins in plants.