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

Silvia Hüttner,Jennifer Schoberer,Ulrike Vavra,Christiane Veit,Daniel Maresch,Richard Strasser,Martina Dicker,Friedrich Altmann

doi:10.1042/bj20141057

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.

Highlights

The endoplasmic reticulum (ER) is the major site for protein folding and maturation in the secretory pathway of eukaryotic cells
STRUBBELIG extracellular domain (SUBEX)-C57Y harbours a signal peptide, the leucine- and proline-rich repeat domains and all three predicted N-glycosylation sites from SUB (Figure 1B) [22]. To monitor their expression and subcellular localization, we generated plant expression constructs, where GFP was fused to the C-terminal end of SUBEX and SUBEX-C57Y and the resulting SUBEX-GFP and SUBEX-C57Y-GFP proteins were transiently expressed in N. benthamiana leaf epidermal cells by agrobacterial infiltration
No colocalization was observed for SUBEX-C57Y-GFP with the Golgimarker MNS1-monomeric RFP (mRFP) [23], indicating that SUBEX-C57Y is exclusively present in the ER (Figure 2D)

Summary

Introduction

The endoplasmic reticulum (ER) is the major site for protein folding and maturation in the secretory pathway of eukaryotic cells. We expressed the glycosylated extracellular domain of SUB-C57Y (called SUBEXC57Y) in Nicotiana benthamiana and A. thaliana plants and found that this misfolded protein is subjected to ERAD in a glycan-dependent manner.

Results

Conclusion