A context-independent N-glycan signal targets the misfolded extracellular domain of Arabidopsis STRUBBELIG to endoplasmic-reticulum-associated degradation

Abstract

N-glycosylation of proteins plays an important role in the determination of the fate of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Specific oligosaccharide structures recruit molecular chaperones that promote folding or mannose-binding lectins that assist in the clearance of improperly-folded glycoproteins by delivery to ER-associated degradation (ERAD). In plants, the mechanisms and factors that recognize non-native proteins and sort them to ERAD are poorly understood. In the present study, we provide evidence that a misfolded variant of the STRUBBELIG (SUB) extracellular domain (SUBEX-C57Y) is degraded in a glycan-dependent manner in plants. SUBEX-C57Y is an ER-retained glycoprotein with three N-glycans that is stabilized in the presence of kifunensine, a potent inhibitor of α-mannosidases. Stable expression in Arabidopsis thaliana knockout mutants revealed that SUBEX-C57Y degradation is dependent on the ER lectin OS9 and its associated ERAD factor SEL1L. SUBEX-C57Y was also stabilized in plants lacking the α-mannosidases MNS4 and MNS5 that generate a terminal α1,6-linked mannose on the C-branch of N-glycans. Notably, the glycan signal for degradation is not constrained to a specific position within SUBEX-C57Y. Structural analysis revealed that SUBEX-C57Y harbours considerable amounts of Glc1Man7GlcNAc2 N-glycans suggesting that the ER-quality control processes involving calnexin/calreticulin (CNX/CRT) and ERAD are tightly interconnected to promote protein folding or disposal by termination of futile folding attempts.

New Insights on Leucine-Rich Repeats Receptor-Like Kinase Orthologous Relationships in Angiosperms

“…The LRR-RLKs are part of the large family of plant proteins which are N-glycosylated and many N-glycosylation acceptor sequences are present in all ECDs. In some pattern recognition receptors and receptors involved in developmental processes, proteins with mutations at residues which will create misfolded proteins have been shown to be part of endoplasmic reticulum protein complexes and directed to degradation (Hong et al, 2008, 2009, 2012; Li et al, 2009; Nekrasov et al, 2009; Lee et al, 2011; Su et al, 2011; Huttner and Strasser, 2012; Sun et al, 2012; Park et al, 2013; Robatzek and Wirthmueller, 2013; Huttner et al, 2014). The significance of all the structural feature modifications which have been mentioned above are still mostly unknown but classic biochemical and cell biological studies (e.g., domain swapping among orthologs and/or targeted point mutations using CRISPR/Cas9) should help to explore their functions in details and will provide many novel insights into the molecular characterization of LRR-RLKs.…”

Review/N-glycans: The making of a varied toolbox

“…SUBEX-Cys57Tyr is an ER-retained glycoprotein with three oligomannosidic N-glycans, but only a single N-glycan is necessary and sufficient for its degradation. This N-glycan signal for degradation is not constrained to a specific position within the protein [119].Also the occupancy of the potential N-glycosylation sites on KOR1 is more critical for correct KOR1 functioning than the structure of the attached N-glycans [126]. KOR1 is an endo-1,4-glucanase involved in the synthesis of cellulose.It is a glycosylated membrane protein with multiple N-glycans that follows the secretory pathway to reach its final destinations in the trans-Golgi network and in the plasma membrane.At least one N-glycan is needed for KOR1 exit from the ER [125].…”

“…Activated EFR induces seedling growth arrest, but os9-1 seedlings that are germinated in the presence of the bacterial peptide elf18 display a retarded growth phenotype similar to wild type plants [120]. is degraded in a glycan-dependent way [119]. SUBEX-Cys57Tyr is an ER-retained glycoprotein with three oligomannosidic N-glycans, but only a single N-glycan is necessary and sufficient for its degradation.…”

“…which has an ERAD-independent function and is involved in N-glycan maturation of completely folded glycoproteins (Figs. 3, 6A) [118][119]. The α1,6-linked mannose residue generated by MNS4 and MNS5is recognized by the ERAD associated mannose-binding lectin AtOS-9 [120].…”

The glycan‐dependent ERAD machinery degrades topologically diverse misfolded proteins

“…In a previous study we have shown that a misfolded variant of the extracellular domain from Arabidopsis STRUBBELIG (SUBEX‐C57Y) is a canonical ERAD substrate that is degraded in a glycan‐dependent manner in plants (Hüttner et al ., ). STRUBBELIG is a leucine‐rich repeat receptor‐like kinase that is crucial for tissue morphogenesis of different plant organs (Vaddepalli et al ., ).…”