Figure 4.

EDEM1 MLD interacts with Z/NHK through Cys²⁵⁶ and possesses reactive thiols. A, MLD-FLAG was coexpressed with A1AT WT, Z, or NHK in HEK293T cells. Cells were radiolabeled for 30 min and chased for 15 min. DTT (5 mm) was added to the cells for 30 min where indicated (+DTT). Cells were lysed in MNT buffer containing TX. A portion of the lysate (65%) was subjected to sequential immunoprecipitation (first (1), anti-FLAG; second (2), anti-A1AT), 20% was subjected to anti-A1AT, and 10% was subjected to anti-FLAG. The proteins were resolved by 8% reducing SDS-PAGE. B, MLD-FLAG was coexpressed with A1AT WT, Z, Z C256S, NHK, NHK C256S, NHK NOG, and NHK NOG C256S in HEK293T cells. Cells were radiolabeled for 30 min, chased for 1 h, and lysed in MNT buffer. Proteins were immunoprecipitated as in A and resolved by 8% reducing SDS-PAGE. The asterisk denotes a background band. C, MLD-FLAG was expressed in HEK293T cells. Cells were pretreated for 1 h with DTT (5 mm) where indicated (+/−). Cells were lysed in sample buffer containing 5 mm PEG-maleimide (+PEG-maleimide) or 20 mm NEM (−PEG-maleimide). Proteins were resolved by 8% SDS-PAGE and immunoblotted (IB) against FLAG antibody. The asterisk denotes a background band. D, MLD-FLAG, MLD C160S/C529S, MLD C410S/C457S, MLD C302S/C555S, and Cys-less MLD were coexpressed in HEK293T cells with WT A1AT, Z, or NHK. The proteins were radiolabeled for 30 min and chased for 1 h. Proteins were immunoprecipitated as in A and resolved by 8% reducing SDS-PAGE. Gels are representative of three independent experiments.