A Novel Stress-induced EDEM Variant Regulating Endoplasmic Reticulum-associated Glycoprotein Degradation

Abstract

Proteins expressed in the endoplasmic reticulum (ER) are subjected to a tight quality control. Persistent association with ER-resident molecular chaperones prevents exit of misfolded or incompletely assembled polypeptides from the ER and forward transport along the secretory line. ER-associated degradation (ERAD) is in place to avoid ER constipation. Folding-incompetent products have to be identified to interrupt futile folding attempts and then targeted for unfolding and dislocation into the cytosol for proteasome-mediated destruction. These processes are better understood for N-glycosylated proteins that represent the majority of polypeptides expressed in the ER. EDEM, a mannosidase-like chaperone, regulates the extraction of misfolded glycoproteins from the calnexin cycle. Here we identify and characterize EDEM2, a novel, stress-regulated mannosidase-like protein that operates in the ER lumen. We show that transcriptional up-regulation of EDEM2 depends on the ER stress-activated transcription factor Xbp1, that EDEM2 up-regulation selectively accelerates ERAD of terminally misfolded glycoproteins by facilitating their extraction from the calnexin cycle, and that the previously characterized homolog EDEM is also a soluble protein of the ER lumen in HEK293 cells.

Protein Folding and Quality Control in the ER

“…EDEM1 presumably possesses mannosidase activity that trims the C branch of N-glycans on misfolded proteins; however, that activity is apparently not required for ERAD acceleration because mutant EDEM1 that lacks the putative active site for mannosidase is still able to accelerate ERAD (Hosokawa et al 2001(Hosokawa et al , 2006(Hosokawa et al , 2010bMolinari et al 2003;Oda et al 2003;Olivari et al 2006). EDEM2 also promotes ERAD, even though it has no enzymatic activity (Mast et al 2005;Olivari et al 2005). In contrast, ERAD-acceleration by EDEM3 (Htm1p/ Mnl1p in yeast) is dependent on its mannosidase activity (Hirao et al 2006;Clerc et al 2009).…”

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation

“…To our surprise, Trichomonas, which makes a Man 5 GlcNAc 2 N-glycan precursor and so was not expected to have N-glycandependent ERAD, contains putative Mns1-like mannosidases, Yos9, and cytosolic PNGases (Figs. 1 E and F and 3B and Table 1) (1)(2)(3)(4)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

“…One model is that Mns1 removes a single Man residue from the middle arm of Man 9 GlcNAc 2 to make Man 8B GlcNAc 2 , which in turn interacts with EDEM that has chaperone and/or lectin activity. This model is complicated by evidence that Mns1 has weak mannosidase activity in S. pombe (17); more than one Man residue may be removed from N-glycans of misfolded proteins in mammalian cells (13); N-glycan-dependent ERAD occurs in mutant cell lines that are missing the middle and upper Man arms on their N-glycans (18); and there are multiple EDEM paralogs, at least two of which (EDEM1 and EDEM3) have mannosidase activity in vivo (19)(20)(21)(22)(23). Yos9, which resembles the Man-6-P receptor, has also been implicated in N-glycandependent ERAD, likely as a lectin (24,25).…”

Intracellular Processing of 1-Antitrypsin

“…The enhanced efficiency of ERAD coincides with the elevated transcription of ER degradation-enhancing mannosidase-like proteins (EDEMs), which are members of the class 47 glycosylhydrolase family (24). Although some EDEM orthologs in lower eukaryotes exhibit mannosidase activity (36), in vitro activity has not been detected for any of the mammalian orthologs (37,38).…”

“…The intracellular degradation of mammalian glycoproteins, via ERAD, involves the removal of several mannose units by ER mannosidase I (ERManI) and perhaps additional members of the class 47 glycosylhydrolase family, especially in yeast (24)(25)(26)(27)(28). The modified glycans promote extraction of glycoproteins from the calnexin cycle (18), and, in combination with nonnative protein structure, are suspected of completing the formation of a proposed bipartite glycoprotein ERAD (GERAD) signal.…”