Mannose trimming is the dominant signal for the release of misfolded glycoproteins from ER quality control

N-glycosylation is essential for protein folding in the endoplasmic reticulum (ER). Glycan attachment facilitates the binding of newly synthesized polypeptides to calnexin and calreticulin, two ER-resident lectins that act as chaperones and promote folding. The regulatory mechanism underlying this process is dictated by the glycan composition, and this study has elucidated the function of mannose trimming in the release of misfolded glycoprotein from ER quality control and subsequent transfer to ER-associated degradation (ERAD) in plants. Coimmunoprecipitation experiments were performed to investigate the interaction between the ERAD substrate SUBEX-C57Y and ER-resident lectins, providing evidence for a specific interaction with calnexin. Furthermore, the results show that the expression of calnexin and calreticulin can specifically inhibit the degradation of glycosylated ERAD substrates. However, overexpression of the α-mannosidase MNS4 was shown to overcome lectin-dependent accumulation and promote degradation via ERAD. MS-based analysis of released N-glycans and jack bean α-mannosidase-digested glycopeptides revealed the presence of monoglucosylated N-glycans even after mannose trimming. This finding is consistent with the observation that calnexin remained associated with SUBEX-C57Y after mannose removal from the C-branch of the N-glycan. In contrast, specific removal of the first α1,2-linked mannose from the A-branch or a block of glucose trimming of Glc3Man9GlcNAc2 prevented the interaction with calnexin. In conclusion, the study provides fundamental insights into the specific role of glycan residues for the interdependence between lectin-based ER quality control and glycan-dependent ERAD in plants.