1. Volume 41, Issue 4, Pages 432-444 (February 2011)
Structural Basis of an ERAD Pathway Mediated by the ER-Resident Protein Disulfide Reductase ERdj5 
Masatoshi Hagiwara, Ken-ichi Maegawa, Mamoru Suzuki, Ryo Ushioda, Kazutaka Araki, Yushi Matsumoto, Jun Hoseki, Kazuhiro Nagata, Kenji Inaba 
Molecular Cell 
Volume 41, Issue 4, Pages (February 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 41, 432-444DOI: (10.1016/j.molcel.2011.01.021)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Primary Structure of Mouse ERdj5
(A) Domain organization of mouse ERdj5 based on the present crystal structure analysis. The CXXC sequences indicate the location of the redox-active sites in the Trx domains (Trx1–Trx4).
(B) Amino acid sequence of mouse ERdj5. Helices and strands are represented by cylinders and arrows, respectively. Active site cysteines located at the N terminus of α2 and a highly conserved proline residue located around the center of α2 in Trx1–Trx4 are in red letters. A redox-active disulfide at a CXXC motif is marked with a black line, while a possibly redox-active cysteine pair in Trxb1 is with a black broken line. Each Trx domain also contains a non-redox-active or structural disulfide bond, which is marked with a blue line. Reverse triangles indicate the cis-proline residue near the redox-active site. The dotted line and the bars above the sequence denote the signal sequence and the domain architecture, respectively.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

4. Figure 2 Overall Structure of ERdj5
(A) Ribbon diagram of ERdj5 with the J, Trx1, Trxb1, Trxb2, Trx2, Trx3, and Trx4 domains in yellow, magenta, light pink, green, blue, red, and orange, respectively. The side chains of the redox-active sites in Trx1–Trx4 are shown in space-filling representation and encircled. Note that the active site cysteines are replaced with serine in this construct.
(B) Ribbon diagram from (A) rotated by 90° around the horizontal axis.
(C) Structural comparison of the Trx domains of ERdj5. Each Trx domain is shown roughly in the same orientation, with the C-terminal α helix (α4) placed at the bottom. The side chains of the redox-active sites in Trx1–Trx4 and a possible redox-active site in Trxb1 are shown in space-filling representation.
(D) Close-up view of the J domain of ERdj5. The partially modeled J domain of full-length ERdj5 is shown in yellow, while the whole J domain derived from J-Trx1 superimposed to Trx1 of full-length ERdj5 is in gray. The ribbon diagram shown in cyan represents the putative whole J domain that is superimposed to the partially modeled J domain of full-length ERdj5. Note that the J domain in full-length ERdj5 is moved away from the predicted position due to the steric hindrance between the α4 helix of the J domain and Trx2 (red arrow). The side chains of the HPD motif in the J domain and the redox-active site in Trx2 are shown in space-filling representation. For clarity, Trx4 of full-length ERdj5 is not shown in this panel.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

5. Figure 3
Domain Organization of other PDI Family Member Proteins with Known Structures
(A) Schematic and three-dimensional structures of yeast PDI (PDB ID: 2B5E) and yeast Mpd1p (PDB ID: 3ED3). The side chains of the CXXC motifs in redox-active Trx domains are shown in space-filling representation and encircled.
(B) Comparison of the domain orientations between ERdj5 and yeast PDI. The N-terminal domain a of PDI and Trx1 of ERdj5 are superimposed, such that the RMSD of their Cα atoms are minimized.
(C) Comparison of the domain orientations between ERdj5 and yeast Mpd1p. The N-terminal domain a of Mpd1p and Trx1 of ERdj5 are superimposed, such that the RMSD of their Cα atoms are minimized.
(D) Surface representations of ERdj5 and yeast PDI. Hydrophobic residues are colored in gray. Regions of basic (>70 kBT/e) and acidic (<–70 kBT/e) potential are blue and red, respectively. The surfaces of the central cleft regions are highlighted in the insets. The molecules in the left panels are shown in roughly the same orientations as in Figure 2A for ERdj5 and in panel (A) for PDI.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

6. Figure 4 Trx3 and Trx4 Domains of ERdj5 Have ERAD-Enhancing Activity
HEK293 cells cotransfected with ERdj5 variants and NHK were labeled for 15 min with [35S] methionine/cysteine 24 hr after transfection and chased for the periods indicated. The metabolically labeled NHK was immunoprecipitated with anti-α1-antitrypsin antibody and subjected to reducing (A–D) or nonreducing (E) SDS-PAGE (upper panel). The band intensities of NHK were plotted in the lower panel. Results presented in the graphs represent the mean ± SD of three independent experiments.
(A) The requirement of the CXXC motifs in the promotion of ERAD by ERdj5. Note that ERdj5/AA does not promote ERAD of NHK.
(B and C) The ERAD-enhancing activities of ERdj5 mutants bearing a single CXXC motif (C1–C4). Note that neither C1 nor C2 accelerates degradation of NHK, whereas both C3 and C4 do.
(D) Different contribution of the N-terminal and C-terminal clusters to ERAD acceleration. Note that C12 does not accelerate ERAD of NHK, whereas C34 does.
(E) Faster decrease of the NHK dimer in the WT ERdj5 or C34 transfected cell than in C12 transfected cell. The band intensities of the NHK dimer were plotted in the lower panel.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 5
Reductase Activity and Redox Potential of ERdj5 Mutants with a Single CXXC Motif
(A) Insulin reduction assays for the ERdj5 mutants with a single CXXC motif (C1–C4).
(B) Redox equilibrium of C1–C4 mutants with glutathione. The oxidized (nonmodified) and reduced (modified) forms of the ERdj5 mutants were separated by SDS-PAGE and stained with CBB. A single asterisk presumably corresponds to a reduced form of ERdj5/AA and C2, in which an unidentified disulfide is reduced and modified with mPEG2000-mal. Double asterisks denote the unusually stable glutathionylated form of C3.
(C) The band intensities in (B) were quantified and redox equilibrium constants of C1, C3, and C4 were calculated by nonlinear fitting (see the Supplemental Experimental Procedures).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

8. Figure 6
Functional Interplay between EDEM1 and the C-Terminal Cluster of ERdj5
(A) Coimmunoprecipitation of EDEM1 with the ERdj5 derivatives in the presence of coexpressed NHK. Immunoprecipitates using an α-FLAG antibody were subjected to reducing SDS-PAGE and analyzed by immunoblotting with α-HA (top). Reversely, immunoprecipitates using an α-HA antibody were subjected to reducing SDS-PAGE and analyzed by immunoblotting with α-FLAG (bottom).
(B) Coimmunoprecipitation of NHK with the ERdj5 derivatives in the presence of coexpressed EDEM1. Immunoprecipitates using an α-FLAG antibody were subjected to reducing SDS-PAGE and analyzed by immunoblotting with an α-A1AT antibody (top). Reversely, immunoprecipitates using an α-A1AT antibody were subjected to reducing SDS-PAGE and analyzed by immunoblotting with α-FLAG (bottom).
(C) Redox state of NHK that was coprecipitated with the ERdj5 derivatives in the presence of coexpressed EDEM1. The cell lysate (top) and immunoprecipitates using an α-FLAG antibody (bottom) were subjected to “nonreducing” SDS-PAGE and analyzed by immunoblotting with an α-A1AT antibody.
(D) Sequential transfer of NHK from calnexin (CNX) to downstream ERAD components. HEK293 cells cotransfected with EDEM1, ERdj5/CA, and NHK were labeled for 9 min with [35S] methionine/cysteine 24 hr after transfection and chased for the periods indicated. The metabolically labeled CNX, EDEM1, ERdj5, and NHK were immunoprecipitated with α-CNX, α-HA, α-FLAG, or anti-α1-antitrypsin antibodies, and the immune complexes were directly subjected to reducing SDS-PAGE. The band intensities of NHK coprecipitated with CNX, EDEM1, or ERdj5 were plotted in the lower panel. Results presented in the graphs represent the mean ± SD of three independent experiments.
(E) Formation of the complex including ERdj5, BiP, and NHK. HEK293 cells cotransfected with EDEM1, ERdj5/CA, NHK, and BiP were labeled for 10 min with [35S] methionine/cysteine 24 hr after transfection and chased for the periods indicated. The metabolically labeled BiP was immunoprecipitated with an α-BiP antibody, and the immune complexes were directly subjected to reducing SDS-PAGE.
(F) Total levels of NHK in HEK293 cells. The metabolically labeled NHK was immunoprecipitated with an α-A1AT antibody, and the immune complex was directly subjected to reducing SDS-PAGE.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

9. Figure 7 Possible Model of the ERdj5-Mediated ERAD Pathway
EDEM1 that is complexed with ERdj5 through the C-terminal cluster recognizes terminally misfolded proteins selectively. The highly reducing C-terminal cluster of ERdj5 then reduces aberrant disulfide bonds of misfolded proteins so that they assume more extended conformation. Reduced and extended substrate polypeptides are likely captured by BiP that binds the J domain of ERdj5 in an ATP-dependent manner and transferred to the retrotranslocation channel upon ATP hydrolysis. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions