IDENTIFYING FUNCTIONAL COMPONENTS OF THE ENDOPLASMIC RETICULUM QUALITY CONTROL AND DEGRADATION FACTOR EDEM1

The ER Degradation-Enhancing Mannosidase-Like protein 1 (EDEM1) is a critical endoplasmic reticulum (ER) quality control factor involved in identifying and directing non-native proteins to the ER-associated protein degradation (ERAD) pathway. However, its recognition and binding properties have remained enigmatic since its discovery. Here we provide evidence for an additional redox-sensitive interaction between EDEM1 and Z/NHK that requires the presence of the single Cys on the α-1 antitrypsin ERAD clients. Moreover, this Cys-dependent interaction is necessary when the proteins are isolated under stringent detergent conditions, ones in which only strong covalent interactions can be sustained. This interaction is inherent to the mannosidase-like domain (MLD) of EDEM1, for which we demonstrate substrate-binding and glycan-trimming properties. EDEM1 appears to possess bipartite binding properties as it also binds these ERAD variants in a Cys-independent manner under milder detergent conditions. In addition, a second intrinsically disordered region (IDR) was identified and both are required for machinery interaction, but they are not necessary for Z/NHK binding. Interestingly, although the N-terminal IDR is required for binding to another ERAD substrate, in this case, neither IDR is required for Z/NHK binding, as the MLD, which lacks both IDRs, exhibits glycan-independent and redox-sensitive binding to Z/NHK. Not only is the MLD sufficient to preferentially interact with the α-1 antitrypsin ERAD clients, it also possesses, as demonstrated for the first time, catalytic properties as observed through glycan-trimming. Although the IDRs do not contribute to Z/NHK binding, they are required for binding to the oxidoreductase, ERdj5. These results expand our understanding of the range of binding interactions implemented by EDEM1 and help elucidate its role as an active mannosidase involved in the quality control machinery of the Endoplasmic Reticulum (ERQC)

EDEM1\u27s mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase–like 1 protein (EDEM1) is a protein quality control factor that was initially proposed to recognize N-linked glycans on misfolded proteins through its mannosidase-like domain (MLD). However, recent studies have demonstrated that EDEM1 binds to some misfolded proteins in a glycan-independent manner, suggesting a more complex binding landscape for EDEM1. In this study, we have identified a thiol-dependent substrate interaction between EDEM1 and the α1-antitrypsin ER-associated protein degradation (ERAD) clients Z and NHK, specifically through the single Cys residue on Z/NHK (Cys256), required for binding under stringent detergent conditions. In addition to the thiol-dependent interaction, the presence of weaker protein-protein interactions was confirmed, suggestive of bipartite client-binding properties. About four reactive thiols on EDEM1 were identified and were not directly responsible for the observed redox-sensitive binding by EDEM1. Moreover, a protein construct comprising the EDEM1 MLD had thiol-dependent binding properties along with its active glycan-trimming activities. Lastly, we identified an additional intrinsically disordered region (IDR) located at the C terminus of EDEM1 in addition to its previously identified N-terminal IDR. We also determined that both IDRs are required for binding to the ERAD component ERdj5 as an interaction with ERdj5 was not observed with the MLD alone. Together, our findings indicate that EDEM1 employs different binding modalities to interact with ERAD clients and ER quality control (ERQC) machinery partners and that some of these properties are shared with its homologues EDEM2 and EDEM3

Analysis of Disulfide Bond Formation

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE