Characterization of Early EDEM1 Protein Maturation Events and Their Functional Implications

Abstract

The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.The signal hypothesis states that proteins are targeted to the eukaryotic secretory pathway by hydrophobic signal sequences that are frequently positioned at the N terminus of the protein (1). These proteins are cotranslationally targeted and translocated across the endoplasmic reticulum (ER) 2 membrane where the vast majority of the maturation process occurs. Maturation events include signal sequence cleavage, protein folding, covalent modification, and assembly (2). The ER contains a number of factors that aid in these steps for secretory and membrane proteins. As protein maturation is an error-prone process, the ER also possesses a quality control process that monitors the successfulness of these steps (3, 4). Quality control factors dedicated to the evaluation and sorting of maturing proteins target defective proteins for destruction by the cytoplasmic proteasome after their dislocation through the ER membrane by a process termed ER-associated degradation (ERAD). Although many of the players involved in ER quality control have been uncovered over the past decade, there is still much to be learned about the mechanism of the quality control process.Signal sequences that target proteins to the ER generally consist of the first 20 -30 amino acids of a protein organized into three domains (5). Signal sequences start with a basic N-terminal domain (N-domain) followed by an extended hydrophobic domain (H-domain; ϳ7-13 amino acids in length) and a polar domain (C-domain) that contains low molecular weight amino acids near the cleavage site. Recent evidence indicates that signal sequences do not solely provide transient targetin...

Arabidopsis Class I α-Mannosidases MNS4 and MNS5 Are Involved in Endoplasmic Reticulum–Associated Degradation of Misfolded Glycoproteins

“…However, we observed that MNS5 can promote the demannosylation of the N-glycan from GFP glyc -HDEL. In mammals, recent data propose the occurrence of soluble and membrane-bound forms for EDEM1, which might have distinct functions in ERAD (Tamura et al, 2011). Whether such variable protein topologies exist for MNS4 and MNS5 remains to be shown in the future.…”

A Golgi-localized Mannosidase (MAN1B1) Plays a Non-enzymatic Gatekeeper Role in Protein Biosynthetic Quality Control

“…Although much of the literature supports the role of mannose trimming from misfolded proteins as critical to their recognition for ERAD, one cannot overlook the fact that much of the ERAD quality control machinery, including EDEM1, OS-9, XTP3-B, and SEL1L, are themselves N-glycosylated. Because the removal of mannose units from their N-glycans is required for ERAD (24,57), one should consider in future studies that treatment with KIF might actually function by inhibiting critical ERAD associations with SEL1L (24,48,49).…”

Glycosylation-directed quality control of protein folding

“…EDEM1 (or just EDEM in earlier reports) has homology to class I α-1,2-mannosidases (which constitute the glycosyl hydrolase family 47). EDEM1 can give rise to soluble and membrane forms because of its inefficiently cleaved ER signal sequence 123,124 . As with ERManI, the role of EDEM1 varies depending on the substrate examined.…”