Figure 1. Effect of mutation of various cysteine residues in EDEM2 on gpERAD.

(A) Structure of Glc₃Man₉GlcNAc₂ is schematically shown. Mannose residues are α1,2-bonded or α1,6-bonded as indicated. (B) Structures of human EDEM2 and yeast Htm1p are schematically shown with cysteine residues (C) highlighted together with their positions (black bars underneath C indicate conserved cysteine residues, whereas white bars over C indicate non-conserved cysteine residues). The purple and yellow boxes denote the signal sequence and mannosidase homology domain (MHD), respectively. Sequence comparison around the four cysteine residues conserved between EDEM2 and Htm1p (marked in red color) is shown below (asterisk and colon indicate identical and similar amino acids, respectively). (C) Cell lysates were prepared from WT and EDEM2-KO cells expressing mCD3-δ-ΔTM-HA by transfection, treated with (+) or without (-) EndoH, and analyzed by immunoblotting using anti-HA antibody. mCD3-δ-ΔTM-HA* denotes deglycosylated mCD3-δ-ΔTM-HA. (D) Cell lysates were prepared from WT and EDEM2-KO cells expressing WT or one of various cysteine mutants of 3x Flag-tagged EDEM2 together with mCD3-δ-ΔTM-HA by transfection, and analyzed by immunoblotting using anti-HA and anti-EDEM2 antibodies. E117Q is an enzymatically inactive mutant of EDEM2. (E) Cycloheximide chase was conducted to determine the degradation rate of mCD3-δ-ΔTM-HA in WT and EDEM2-KO cells expressing WT or one of the three cysteine mutants of 3x Flag-tagged EDEM2 by transfection, and analyzed by immunoblotting using anti-HA antibody (n = 3). Quantified data are shown on the right.