Abstract
Protein folding in the cell is regulated by several quality-control mechanisms. Correct folding of glycoproteins in the endoplasmic reticulum (ER) is tightly monitored by the recognition of glycan signals by lectins in the ER-associated degradation (ERAD) pathway. In mammals, mannose trimming from N-glycans is crucial for disposal of misfolded glycoproteins. The mannosidases responsible for this process are ER mannosidase I and ER degradation-enhancing α-mannosidase-like proteins (EDEMs). However, the molecular mechanism of mannose removal by EDEMs remains unclear, partly owing to the difficulty of reconstituting mannosidase activity in vitro Here, our analysis of EDEM3-mediated mannose-trimming activity on a misfolded glycoprotein revealed that ERp46, an ER-resident oxidoreductase, associates stably with EDEM3. This interaction, which depended on the redox activity of ERp46, involved formation of a disulfide bond between the cysteine residues of the ERp46 redox-active sites and the EDEM3 α-mannosidase domain. In a defined in vitro system consisting of recombinant proteins purified from HEK293 cells, the mannose-trimming activity of EDEM3 toward the model misfolded substrate, the glycoprotein T-cell receptor α locus (TCRα), was reconstituted only when ERp46 had established a covalent interaction with EDEM3. On the basis of these findings, we propose that disposal of misfolded glycoproteins through mannose trimming is tightly connected to redox-mediated regulation in the ER.
Highlights
Protein folding in the cell is regulated by several quality-control mechanisms
To identify proteins that regulate the mannose-trimming activity of EDEM3, we first searched for proteins that associate with EDEM3
Because this was reminiscent of the interaction between Htm1p/Mnl1p and Pdi1p [29, 30], we investigated whether EDEM3 binds other protein-disulfide isomerase (PDI) family members
Summary
ERp46 associates with EDEM3 and promotes the mannosetrimming activity of EDEM3 in vivo. In cells co-expressing the CXXA mutant, all of the EDEM3 formed disulfide-bonded complexes, which were detected by SDS-PAGE under nonreducing conditions (Fig. 2F, lane 7, open arrowheads). In WT ERp46-expressing cells, similar disulfide-bonded complexes were detected in addition to the EDEM3 monomer (Fig. 2F, lane 6), indicating that the interaction of ERp46 with EDEM3 depends on the redox-active sites of ERp46. The covalent complex was detected only in lysates from WT EDEM3-expressing cells resolved under nonreducing conditions (Fig. 3, E and F, open arrowheads) These data suggest that ERp46 forms a disulfide bond between Cys and Cys442 in the ␣-mannosidase domain of EDEM3. The amounts of recombinant proteins in the reactions were confirmed by CBB staining (Fig. S8) Taken together, these results strongly suggest that the covalent interaction of ERp46 with EDEM3 triggers mannose trimming of EDEM3
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