RESEARCH ARTICLE

Mechanistic characterization of disulﬁde bond reduction of
an ERAD substrate mediated by cooperation between ERdj5
and BiP
Received for publication, March 10, 2023, and in revised form, August 28, 2023 Published, Papers in Press, September 21, 2023,
https://doi.org/10.1016/j.jbc.2023.105274

Xiaohan Cai1,2 , Shogo Ito1,2, Kentaro Noi3, Michio Inoue1, Ryo Ushioda4 , Yukinari Kato5, Kazuhiro Nagata4, andKenji Inaba1,2,6,7,8, *From the 1Institute of Multidisciplinary Research for Advanced Materials, and 2Department of Molecular and Chemical Life
Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; 3Department of Biotechnology and Life
Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan; 4Faculty of Life Sciences, Kyoto Sangyo
University, Kyoto, Japan; 5Graduate School of Medicine, Tohoku University, Sendai, Japan; 6Department of Chemistry, Graduate
School of Science, Tohoku University, Sendai, Miyagi, Japan; 7Medical Institute of Bioregulation, Kyushu University, Fukuoka,
Japan; 8Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development(AMED), Tokyo, Japan
Reviewed by members of the JBC Editorial Board. Edited by Wolfgang Peti

Endoplasmic reticulum (ER)-associated degradation (ERAD)
is a protein quality control process that eliminates misfolded
proteins from the ER. DnaJ homolog subfamily C member 10(ERdj5) is a protein disulﬁde isomerase family member that
accelerates ERAD by reducing disulﬁde bonds of aberrant
proteins with the help of an ER-resident chaperone BiP.
However, the detailed mechanisms by which ERdj5 acts inconcert with BiP are poorly understood. In this study, wereconstituted an in vitro system that monitors ERdj5-mediatedreduction of disulﬁde-linked J-chain oligomers, known to bephysiological ERAD substrates. Biochemical analyses using
puriﬁed proteins revealed that J-chain oligomers were reduced
to monomers by ERdj5 in a stepwise manner via trimeric anddimeric intermediates, and BiP synergistically enhanced thisaction in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using highspeed atomic force microscopy, demonstrated the stochastic
release of small J-chain oligomers through repeated actions of
ERdj5 on peripheral and ﬂexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected
at the molecular level.

Secretory proteins, translocated across the endoplasmic reticulum (ER) membrane in an unfolded form (1), fold with thehelp of ER-resident molecular chaperones, protein disulﬁdeisomerase family members, N-glycosylation enzymes, andvarious other proteinaceous and chemical factors. Stringent
cellular quality control systems allow only correctly folded
proteins to exit the ER for transport to their ﬁnal destinations(2). By contrast, misfolded proteins tend to accumulate in the
ER and induce severe ER stresses, resulting in harmful effects
on living organisms. This can result in a variety of diseases* For correspondence: Kenji Inaba, kenji.inaba.a1@tohoku.ac.jp.

such as Alzheimer’s disease (3), Parkinson’s disease (4), andtype-2 diabetes (5). To maintain ER proteostasis, misfolded
proteins are retrogradely delivered from the ER to the cytosol
to be degraded by proteasomes in a process called ERassociated degradation (ERAD) (6, 7). During this process,disulﬁde bond reduction of misfolded proteins is likely acritical step that facilitates their passing through the dislocon
channel, thereby promoting their ERAD without pausing (8).
DnaJ homolog subfamily C member 10 (ERdj5) was
discovered as a protein disulﬁde isomerase family memberthat catalyzes disulﬁde reduction of misfolded proteins in theoxidative environment of the ER and accelerates their ERAD
(9, 10). Misfolded “glycoproteins” are ﬁrst recognized by ER
degradation–enhancing mannosidase-like protein 1 (11)and reduced by ERdj5 via recruitment of ERdj5 by ER
degradation–enhancing mannosidase-like protein 1 (12). The
reduced proteins are then delivered to the dislocon channel(Sec61) by immunoglobulin-binding protein (BiP), translocated to the cytoplasm, and eventually degraded by theubiquitin-proteasome system (13). On the other hand, misfolded “non-glycoproteins” are ﬁrst recognized by BiP andthen transferred to ERdj5 via speciﬁc interactions between the
ERdj5 J-domain and BiP (14). In either case, the cooperationof ERdj5 and BiP is believed to be essential for enhancing
ERAD.
Our previous crystallographic studies revealed that ERdj5
consists of N-terminal and C-terminal clusters and that these
two are connected by a ﬂexible linker loop (10, 15). In total,
there are six tandem thioredoxin (Trx)-like domains in ERdj5,
four (Trx1−4 domain) of which have a CXXC motif at theredox-active site. While the N-terminal cluster contains Trx1,
Trx2, and two redox-inactive Trx domains, the C-terminal
cluster is composed of Trx3 and Trx4 domains. The redox
potential of each Trx-like domain has been determined tobe −166 mV for Trx1 domain, −172 mV for Trx3 domain,and −185 mV for Trx4 domain. Notably, the CXXC motif of
J. Biol. Chem. (2023) 299(11) 105274

1© 2023 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC
BY license (http://creativecommons.org/licenses/by/4.0/).

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
Trx2 domain is masked by the J-domain, preventing substratemolecules from accessing this redox-active site. As a consequence, the reducing ability of Trx2 domain is much weaker
than that of the other Trx-like domains. Mutational analyses
further demonstrated that Trx3 domain and Trx4 domain in
the C-terminal cluster are the primary reductase domains
responsible for the ERAD-enhancing activity of ERdj5 (10),while the N-terminal J-domain with a His-Pro-Asp (HPD)
motif is essential for binding BiP and stimulating its ATPase
activity (16). More recently, another crystal structure wasdetermined for ERdj5, in which the relative orientation andlocation of the C-terminal cluster to the N-terminal one differ
largely from those in the original structure (15). High-speed
atomic force microscopy (HS-AFM) measurements revealed
that these two different conformations are in equilibrium,concomitant with the rapid movement of the C-terminal
cluster (15).
While physiological and biochemical studies have also since
been conducted to investigate roles of ERdj5 as a disulﬁdereductase (17–19), detailed mechanisms of the ERdj5-BiP
interplay during the disulﬁde reduction of ERAD substrates
remains poorly understood. In this study, we set up an in vitro
experimental system that monitors J-chain oligomer disaggregation mediated by the cooperation of ERdj5 and BiP. Jchain, a protein component of immunoglobulins IgM and IgA(20), contains eight cysteine residues and has a molecular massof 25 kDa per monomer. It forms disulﬁde-linked oligomers
when overexpressed alone in cells, and the oligomers undergo
ERAD in an ERdj5-dependent manner (9, 15). The present
in vitro analysis using puriﬁed proteins enabled us to follow
the kinetics of J-chain oligomer disassembly mediated by
ERdj5 and BiP and demonstrated that BiP synergistically accelerates ERdj5-catalyzed disulﬁde reduction via generation oftrimeric and dimeric intermediate species. In support of this,real-time, single-molecule observations using HS-AFM
demonstrated that small J-chain fragments were released
from the large J-chain oligomers through repeated attacks of
ERdj5 on peripheral and ﬂexible regions of the oligomers.
Systematic mutational analyses provided further mechanistic
insights into ERAD substrate reduction mediated by thecooperation of ERdj5 and BiP.

by adding puriﬁed ERdj5 (0.5 μM) and/or BiP (0.5 μM) to Jchain oligomer (5 μM; 10-fold excess to enzymes) solution in
the presence of 2.5 mM GSH and 1 mM ATP and was stopped
by addition of 10 mM N-ethylmaleimide (NEM) at different
timepoints. The human BiP protein used in this study retained
ATPase activity, which was enhanced by ERdj5 (Fig. S2).
Whereas GSH alone hardly reduced J-chain oligomers(Fig. 1B), ERdj5 signiﬁcantly promoted their reduction tomonomers (Fig. 1C). Importantly, a small amount of trimer
and larger amount of dimer were generated during the earlystages of J-chain reduction and thereafter both decreased
gradually (at 45 and 60 min). Meanwhile, monomeric J-chain
was greatly increased in the latter stages of the reaction, suggesting the stepwise reduction and conversion of J-chain
oligomers to monomers via trimer and dimer species (Fig. 1, C
and G). The monomer species consisted of oxidized andreduced forms, with the reduced form predominating(Fig. 1C), suggesting that ERdj5 could reduce both intermolecular and intramolecular disulﬁde bonds of J-chains. In the
reducing SDS-PAGE, the J-chain molecules were all converted
to the dimer and monomer species (Fig. S1B). Notably, thesequential reduction of the PA-tagged J-chain oligomers to thetrimer, dimer, and monomer was further veriﬁed by theWestern blotting analysis using an anti-PA antibody (Fig. S3).
However, given that different oligomeric states of proteins may
show different membrane transfer efﬁciencies and/or different
reactivity with an antibody used, we used the Coomassie
brilliant blue (CBB)-staining data for the quantitative analysis
throughout this work.
BiP itself does not contain redox-active sites, hence nomonomer species were generated upon addition of BiP without
GSH (Fig. S1C). However, BiP only generated a small amountof J-chain dimers and monomers in the presence of 2.5 mM
GSH (Fig. 1, D and G). Notably, when both ERdj5 and BiP wereadded to the reaction solution, J-chain oligomers were reduced
to monomers more efﬁciently than expected from the simple
sum of the activities of ERdj5 and BiP (Fig. 1, E, G, and H),
indicating that they act synergistically to accomplish efﬁcient Jchain reduction. No synergy was observed in the absence ofATP (Fig. 1, F–H), suggesting that the ATPase activity of BiP isrequired for this action.

Results
J-chain reduction mediated by ERdj5 and/or BiP

Mechanisms of BiP-dependent acceleration in ERdj5-catalyzed
J-chain reduction

To analyze the ERdj5-mediated disulﬁde bond reduction of
an ERAD substrate, we reconstituted the J-chain oligomer
reduction system using nonreducing SDS gels and puriﬁed
ERdj5, BiP, and J-chain oligomers (Fig. 1A). GSH was added asa source of reducing power for ERdj5 (21). Given that GSH
itself may have the potential to reduce J-chain oligomers, andthat GSH is abundant in the ER, ranging from 1 to 10 mM(22), we ﬁrst veriﬁed that 2.5, 5, and 10 mM GSH hardly
reduced J-chain oligomers even after 60 min of reaction time
(Fig. S1A). To minimize the effect of GSH itself and assess thenet contribution of ERdj5 in J-chain reduction, we chose
2.5 mM GSH for this assay system. The reaction was initiated

BiP is abundant in the ER as a primary chaperone that can
respond to ER stress (23). To investigate the role of BiP indisaggregation of J-chain oligomers, we used a higher concentration (2 μM) of BiP in the J-chain reduction assay. At
2 μM, BiP enhanced the generation of J-chain dimers andmonomers with GSH (Fig. 2A); after 60 min, 3-fold moremonomer was generated by 2 μM BiP than by 0.5 μM BiP(Fig. 2E). In the presence of 0.5 μM ERdj5 and 2 μM BiP,
J-chain oligomers were converted to monomers much more
rapidly (Fig. 2, B and D). However, the efﬁciency was almost
the same as that of the combination of 0.5 μM ERdj5 and0.5 μM BiP (Fig. 2E), indicating that excess BiP was unable to2 J. Biol. Chem. (2023) 299(11) 105274

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
A

D

B

C

E

F

G

H

Figure 1. J-chain reduction mediated by ERdj5 and/or BiP. A, puriﬁed ERdj5, BiP, and J-chain oligomer. Proteins were separated by nonreducing SDSPAGE and stained with CBB-G250. B–F, 5 μM J-chain oligomers were reacted with 2.5 mM GSH, 0.5 μM ERdj5, and/or 0.5 μM BiP in the presence of 1 mM ATP(without ATP in F) at 30  C for the speciﬁed time. Reactions were quenched with NEM at the indicated timepoints, and 4 μl of the reaction solution was
subjected to nonreducing SDS-PAGE and stained with CBB G-250. G, band intensity ratio of J-chain monomers (red), dimers (magenta), and trimers (green)
versus total J-chain on each nonreducing SDS-PAGE gel was plotted as a function of reaction time. Results are means ± SD of three independent experiments. H, band intensity ratio of J-chain monomer versus total J-chain on nonreducing SDS-PAGE gels plotted as a function of reaction time based on
the results shown in (B–F). Bip, binding protein; CBB, Coomassie brilliant blue; NEM, N-ethylmaleimide.

J. Biol. Chem. (2023) 299(11) 105274

3

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP

Figure 2. Effects of different concentrations of BiP and their preincubation on ERdj5-mediated J-chain reduction. A and B, 5 μM J-chain oligomers
were reacted with 2 μM BiP, 2.5 mM GSH, and 1 mM ATP in the absence or presence of 0.5 μM ERdj5 for the indicated time at 30  C. Reaction solutions
quenched with NEM at the indicated timepoints were subjected to nonreducing SDS-PAGE and stained with CBB G-250. C and D, band intensity ratio of
J-chain monomers (red) and dimers (magenta) versus total J-chain on each nonreducing SDS-PAGE gel was plotted as a function of reaction time based on
the results shown in (A and B). E, band intensity ratio of J-chain monomers versus total J-chain on each nonreducing SDS-PAGE gel was plotted as a functionof reaction time based on the results shown in (A, B, and Fig. 1, D and E, and F–H). 5 μM J-chain oligomers were preincubated with 0, 0.5, or 2 μM BiP for
30 min before being mixed with 0.5 μM ERdj5 and 2.5 mM GSH in the presence of 1 mM ATP for the indicated time at 30  C. Reaction solutions quenchedwith NEM at the indicated timepoints were subjected to nonreducing SDS-PAGE and stained with CBB G-250. Lane N, no preincubation with GSH, and BiP. I
and J, band intensity ratio of J-chain monomer versus total J-chain (I) and dimer versus total J-chain (J) on nonreducing SDS-PAGE gels plotted as a functionof reaction time. Results are means ± SD of three independent experiments. Bip, binding protein; CBB, Coomassie brilliant blue; NEM, N-ethylmaleimide.

4 J. Biol. Chem. (2023) 299(11) 105274

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
enhance further the reduction of J-chain oligomers. This result
suggests that stoichiometric interplay between ERdj5 and BiP
enables their synergistic actions for J-chain reduction in which
the BiP-recruited substrate can be efﬁciently transferred to andreduced by ERdj5 via speciﬁc interactions between BiP and
ERdj5. In this case, residual BiP would function as a molecularchaperone to disaggregate J-chain oligomers, but this effectseems marginal compared with the cooperative effect of ERdj5
and BiP.
Nonetheless, to assess the function of BiP as a molecularchaperone that promotes disaggregation, we explored the effect of BiP preincubation on ERdj5-mediated J-chain reduction. To this end, J-chain oligomers were preincubated with 0.5
or 2 μM BiP for 30 min and then reacted with 0.5 μM ERdj5(Fig. 2, F–J). In this case, preincubation with BiP greatly
increased the amount of monomer and dimer species, especially at early timepoints of the reaction (0–20 min) and in amanner dependent on the BiP concentration (Fig. 2, G–J).
While preincubation with 0.5 μM BiP yielded monomer species representing 40% of the total after 20 min, the yield wasincreased to 55% of the total following preincubation with
2 μM BiP (Fig. 2I). Thus, BiP can serve as an effective facilitatorof the subsequent ERdj5-mediated disulﬁde reduction. BiP
performs dual roles, acting as general chaperone that disaggregates aberrant oligomers, and as a speciﬁc partner that
recruits substrates to ERdj5, and its role likely changes
depending on the stage at which it participates in the ERdj5dependent ERAD pathway.
To further verify the importance of ERdj5-BiP interplay inJ-chain oligomer reduction, we prepared an ERdj5 mutant
defective in its interaction with BiP. ERdj5 is known to interact
with and activate BiP through the HPD motif located in the
J-domain (9). Accordingly, an H63A mutation at this motif ispredicted to abolish the ability of ERdj5 to activate BiP. Indeed,
the ATPase activity of BiP was enhanced by ERdj5 WT, but
not by the H63A mutant (Fig. S2). The J-chain reduction assay
revealed that the H63A mutant retained the ability to reduce
J-chain oligomers to monomers as efﬁciently as WT ERdj5(Fig. 3B). Interestingly, however, BiP did not further accelerate
J-chain reduction mediated by the ERdj5 H63A mutant(Fig. 3D). The increase of a BiP concentration to 2 μM did notenhance the catalysis of ERdj5 H63A (Fig. S4, A and B),
consistent with the observation that this mutant was unable toenhance the BiP ATPase activity at this BiP concentration

Figure 3. J-chain reduction mediated by the ERdj5 J-domain mutant. A, simpliﬁed cartoon representing the ERdj5–BiP interaction through the HPD
motif of the ERdj5 J-domain and the domain organization of ERdj5 H63A (B and C), 5 μM J-chain oligomers were reacted with 0.5 μM ERdj5 H63A, 2.5 mM
GSH, and 1 mM ATP in the absence or presence of 0.5 μM BiP for the indicated time at 30  C. Reaction solutions quenched with NEM at the indicatedtimepoints were subjected to nonreducing SDS-PAGE and stained with CBB G-250. D, band intensity ratio of J-chain monomer versus total J-chain on
nonreducing SDS-PAGE gels plotted as a function of reaction time. Results are means ± SD of three independent experiments. Bip, binding protein; CBB,
Coomassie brilliant blue; HPD, His-Pro-Asp; NEM, N-ethylmaleimide.

J. Biol. Chem. (2023) 299(11) 105274

5

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP(Fig. S4C). These results indicate the essentiality of the functional interplay between ERdj5 and BiP in accelerated J-chain
reduction by ERdj5. In other words, BiP can further promote
ERdj5-catalyzed J-chain reduction by forming an activatedcomplex with ERdj5 via the HPD motif.
Distinct roles of ERdj5 Trx-like domains in J-chain reduction
Our previous studies demonstrated that Trx3 domain andTrx4 domain present in the C-terminal cluster are redoxactive domains essential for ERAD acceleration in cells (10).
To further investigate the roles of Trx3 domain and Trx4
domain in disulﬁde reduction of misfolded proteins, we prepared a set of ERdj5 mutants (C1−C4) in which only one
CXXC motif was retained, while the other three were
substituted by SXXS (Fig. 4A). After overexpression in
HEK293T cells and puriﬁcation (Fig. 4B), the mutant proteinsAC

were used in the J-chain reduction assay. The results demonstrated that the ERdj5 C4 mutant was the most efﬁcient in
reducing J-chain oligomers to monomers, although its efﬁciency was slightly lower than that of the WT. C1 and C3
reduced J-chain oligomers to monomers to a lesser extent than
C4. C2 displayed the lowest reductase activity, consistent with
our structural observation that the redox-active site in Trx2
domain is masked by the N-terminal J-domain of ERdj5 (10).
The above results demonstrated that Trx4 domain possesses
strong reductase activity against J-chain oligomers (10),whereas Trx3 is inferior as a reductase domain to Trx4. The
difference between the reductase activities of Trx4 domain andTrx3 domain could be due to the alternating domain architecture of ERdj5. Our previous crystallographic studies
revealed two different ERdj5 conformations with different
domain arrangements; form-1 and form-2 (15). In form-1, the
CXXC motifs in Trx1, Trx3, and Trx4 domains are exposed toBD

Figure 4. J-chain reduction mediated by ERdj5 Trx-like domain mutants. A, domain organization of ERdj5 based on the present crystal structure
analysis. The CXXC sequences indicate the locations of redox-active sites in Trx domains. B, puriﬁed ERdj5 Trx-like domain mutants. Proteins were separated
by nonreducing SDS-PAGE and stained with CBB-G250. C, 5 μM J-chain oligomers were reacted with 2.5 mM GSH and 0.5 μM ERdj5 Trx-domain mutants inthe presence of 1 mM ATP for the indicated time at 30  C. Reaction solutions quenched with NEM at the indicated timepoints were subjected tononreducing SDS-PAGE and stained with CBB G-250. D, band intensity ratio of J-chain monomer versus total J-chain on nonreducing SDS-PAGE gels plotted
as a function of reaction time in (C). Results are means ± SD of three independent experiments. Data were analyzed by one-way ANOVA and Tukey tests.
Statistically signiﬁcant differences between each ERdj5 derivative are indicated by asterisks (***p < 0.001; ****p < 0.0001). CBB, Coomassie brilliant blue;
NEM, N-ethylmaleimide; Trx, thioredoxin.

6 J. Biol. Chem. (2023) 299(11) 105274

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
bulk solvent, whereas in form-2, the CXXC motif in Trx3
domain is directed toward the inside of the molecule (Fig. 5A).
Thus, Trx3 domain seems unlikely to function as an effective
reductase domain in form-2.
To explore the importance of the alternating conformationsof ERdj5 in efﬁcient J-chain reduction, we prepared the ERdj5
S557P mutant, which was previously shown to adopt form-1
exclusively and constitutively (15). The S557P mutant
reduced J-chain oligomers to monomers, but its efﬁciency was
only slightly lower than that of WT ERdj5 (Fig. 5, B and C). We
also constructed and puriﬁed the S557P C4 mutant, which
retained a redox-active CXXC motif only in Trx4 domain in
the S557P mutant. This mutant reduced J-chain oligomers tomonomers at almost the same rate as ERdj5 S557P. Thus, Trx4
domain retained high J-chain reduction activity even when
ERdj5 was ﬁxed in form-1, suggesting that the reductase activity of Trx4 domain does not rely on the alternating domain
arrangements of ERdj5. By contrast, the contribution of theother Trx-like domains seems negligible, as far as ERdj5
maintains a form-1 conformation.
HS-AFM observation of ERdj5 acting on J-chain oligomers
To directly visualize the action of ERdj5 on J-chain oligomers at the single-molecule level, we employed HS-AFM(24, 25). For this, target molecules are immobilized onto amica surface, and their three-dimensional structure andFigure 5. J-chain reduction mediated by the ERdj5 conformation-ﬁxed mutant. A, crystal structure of ERdj5 S557P (form-1, PDB: 5AYK) and ERdj5 CC(form-2, PDB: 5AYL). The CXXC motifs in Trx1, Trx2, Trx3, and Trx4 domains are circled in green (exposed to surface) or red (concealed). B, 5 μM J-chain
oligomers were reacted with 2.5 mM GSH and 0.5 μM ERdj5 form-1 mutants in the presence of 1 mM ATP for the indicated time at 30  C. Reaction solutions
quenched with NEM at the indicated timepoints were subjected to nonreducing SDS-PAGE and stained with CBB G-250. C, band intensity ratio of J-chain
monomer versus total J-chain on nonreducing SDS-PAGE gels plotted as a function of reaction time. Results are means ± SD of three independent experiments. CBB, Coomassie brilliant blue; NEM, N-ethylmaleimide; Trx, thioredoxin.

J. Biol. Chem. (2023) 299(11) 105274

7

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
dynamics are analyzed by rapidly scanning the surface using acantilever arm with an extremely sharp tip. After numerous
attempts under various conditions, we optimized the experimental conditions for HS-AFM measurement. Brieﬂy, sample
solution containing 100 nM J-chain oligomers and 2.5 mM
GSH was loaded onto a cobalt-scattered mica surface. After
J-chain molecules were immobilized, the surface was washedto remove unbound J-chain molecules, and images were acquired. Based on the crystal structure of a J-chain monomer(26) (Fig. 6A), its N-wing domain is rich in β-strand elements,
whereas the C-wing segment is intrinsically disordered.
Indeed, our HS-AFM images visualized the N- and C-wings ofa J-chain monomer generated as a minor species after an
80 min incubation with 2.5 mM GSH and no ERdj5 (Fig. 6B).
Based on the overall shape and dimension, J-chain monomers,
dimers, and higher-order oligomers were roughly identiﬁedusing this approach (Fig. 6B). Consistent with the above
biochemical assay (Fig. 1B), most of the J-chain molecules
maintained oligomeric states in the presence of 2.5 mM GSH,
even after HS-AFM observation. These observations conﬁrm
that HS-AFM is a useful tool for following the disassembly ofJ-chain oligomers during ERdj5-catalyzed disulﬁde reduction.
Next, we performed HS-AFM observation for the ERdj5mediated J-chain reduction (Fig. 6C). To this end, 10 nM
ERdj5 was added to the cantilever holder to initiate the
J-chain reduction on the mica surface, and the data acquisition was started immediately. Surface coating with Co2+
prevented the adherence of excess ERdj5 molecules. To
conﬁrm the overall shapes of ERdj5 molecules and thereby
distinguish them from the J-chain molecules, we ﬁrst performed the HS-AFM measurements for ERdj5 alone.
Consistent with previous observations (15, 25), we observedthat ERdj5 molecules adopt multiple conformations withdifferent orientations between the N- and C-terminal clusters (Fig. S5).
Figure 6C shows a typical set of HS-AFM images, demonstrating the disassembly of J-chain oligomers upon ERdj5 action. Time-resolved snapshots revealed that an ERdj5 molecule
attacked a peripheral region of the J-chain oligomer at 11.0 s
and thereafter a part of the J-chain oligomer was released at
11.1 s (Fig. 6C). Similar events were observed at different regions of the J-chain oligomer at 46.7 s and 57.5 s, which
eventually resulted in J-chain oligomers of smaller sizes as seen
in snapshots at 47.0 s and 57.7 s, respectively (Fig. 6C). We
additionally observed that an ERdj5 molecule attacked a core
part of the J-chain oligomer from the downside at 50.9 and
51.0 s (Fig. 6C). However, no J-chain fragments were releasedin this case. Collectively, it is suggested that the ERdj5mediated disassembly of the J-chain oligomers tends tooccur when the enzyme attacks peripheral and/or ﬂexible regions of the J-chain oligomers.
To distinguish ERdj5 particles from the J-chain fragments
leaving from the complexes with certainty and thereby interpret the HS-AFM data objectively, we calculated the areas andcircularities of particles observed in HS-AFM images (25, 27,
28). The area versus circularity plot classiﬁed randomly chosen
ERdj5 particles into two major groups, round O-shape (form-

8 J. Biol. Chem. (2023) 299(11) 105274

1) and extended V-shape (form-2) (Fig. 6A left and Fig. S6B),
consistent with our previous crystallographic and HS-AFM
studies (Figs. 5A and S5A) (15). The HS-AFM observation of
J-chain alone in the presence of GSH visualized its dimer andmonomer species located nearby the higher-order J-chain
oligomers (Fig. S6C), showing much wider distribution in area
than ERdj5 on the plot (Fig. S6A, middle). Concretely, whereas
the areas of the ERdj5 molecules distribute mostly around
150 nm2 (Fig. S6A, left), a major portion of the J-chain particles
show the area of less than 100 nm2 while a minor portion
displaying the area of higher than 150 nm2 (Fig. S6A, middle).
Such different distributions on the area versus circularity plots
between the ERdj5 and J-chain particles enabled us to distinguish these two in the HS-AFM images of their mixtures withcertainty (Fig. S6A, right).
Through repeated HS-AFM measurements, 106 J-chain
oligomer molecules were observed, of which 62 were eventually disassembled or detached from the surface. During observations, some of the J-chain oligomers were transiently
bound to multiple ERdj5 molecules at different sites until they
were partially or fully disassembled (Fig. S7). We also observedthat the same ERdj5 molecule attacked the same or similarsites of a J-chain oligomer repeatedly. In either case, ERdj5
molecules preferentially acted on the peripheral and ﬂuctuating regions of J-chain oligomers rather than on their central
core part. We measured the duration of ERdj5 binding toJ-chain oligomers for two hundred ERdj5 molecule. Most
ERdj5 molecules bound J-chain oligomers for <1.5 s per
attack, irrespective of whether target J-chain oligomers wereresolved or not by attack of the ERdj5 molecule (Fig. 6D).
Thus, ERdj5 acts transiently on J-chain oligomers in mostcases and subsequent J-chain disassembly via disulﬁdereduction seems likely to occur through repeated attacks by
ERdj5 in a stochastic manner.

Discussion
In this study, we successfully monitored the sequential
reduction of J-chain oligomers catalyzed by the cooperation of
ERdj5 and BiP. The results revealed that ERdj5 reduces J-chain
oligomers in a stepwise manner via formation of trimeric anddimeric intermediates, while BiP alone resolves J-chain oligomers only partially in the presence of GSH. Importantly, BiP
synergistically accelerated ERdj5-mediated J-chain reduction
in an ATP-dependent manner (Fig. 1), and its functional
interaction with the ERdj5 J-domain was essential for thisacceleration (Fig. 3, C and D). BiP molecules activated by
ERdj5 via its HPD motif may physically loosen aggregatedparts of J-chain oligomers in advance and thereby increase theaccessibility of ERdj5 and GSH to aberrant disulﬁde bonds.
Excess BiP did not further enhance the ERdj5-catalyzed disulﬁde reduction (Fig. 2E), suggesting that the above events
seem likely to be promoted in a 1:1 complex between ERdj5
and BiP. This mechanism appears to be consistent with apreviously proposed model in which misfolded nonglycoproteins such as J-chain oligomers are ﬁrst recognized by
BiP and transferred to an ERdj5 molecule complexed with BiP

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
A

B

C

D

Figure 6. HS-AFM observation of ERdj5 acting on J-chain oligomers. A, structure of the J-chain (monomer) based on cryo-EM analysis of IgM (PDB:
6KXS). Fcμ and J-chain are colored blue and orange, respectively. J-chain has a two-winged structure and interacts with the tailpieces of the Fcμ pentamer. B,
high-speed atomic force microscopy (HS-AFM) images of J-chain molecules differing in size and compactness. 100 nM J-chain was used for immobilization
onto the cobalt-scattered mica surface. After 80 min incubation with 2.5 mM GSH, J-chain molecules were mostly observed as higher-order oligomers, while
the minor portion as monomers and dimers. C, a typical set of HS-AFM snapshots showing that ERdj5 molecules bound to the peripheral regions of the Jchain oligomer, eventually leading to its disaggregation. J-chain oligomers, ERdj5 molecules, and leaving J-chain fragments are indicated by red, blue, andgreen arrows, respectively. D, histogram of the binding time between ERdj5 molecules and J-chain oligomers. Samples (n = 200) were randomly selectedfrom particles that were repeatedly attacked by ERdj5 and eventually disaggregated on the mica surface.

J. Biol. Chem. (2023) 299(11) 105274

9

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
in an ATP-dependent manner to accelerate disulﬁde reduction
and ERAD of the substrates (10).
Our previous studies revealed that the C-terminal cluster of
ERdj5 contains the primary reductase domains, and the highly
dynamic nature of the N- and C-terminal clusters is key to
efﬁcient disulﬁde bond reduction of ERAD substrates (10, 15).
The present biochemical experiments using puriﬁed proteins
veriﬁed that the C-terminal Trx4 domain of ERdj5 plays apivotal role in catalyzing disulﬁde reduction of J-chain oligomers and that limited cluster dynamics led to compromised
J-chain reduction by ERdj5 (Fig. 5).
HS-AFM facilitated visualization of the actions of ERdj5 on
J-chain oligomers at the single-molecule level. We found thatERdj5 molecules preferentially attacked the peripheral andﬂexible regions of J-chain oligomers rather than their central
core parts. In most cases, ERdj5 bound the J-chain oligomers
transiently and disassembled them by cleaving off small J-chain
fragments. Thus, the enzyme is unlikely to bisect J-chain oligomers to nearly half-size fragments. These HS-AFM observations seem consistent with the results of J-chain reduction
assays: J-chain oligomers appear to disassemble sequentially via
the generation of trimeric and dimeric intermediates (Fig. 1).
It will be interesting to explore how many reductive reactions can occur during one binding or collision event. To
calculate the frequency of disulﬁde bond reduction by one
collision of ERdj5 with J-chain oligomers, the exact number ofintermolecular disulﬁde bonds in a higher-order J-chain oligomer and the average interval time as well as the duration of
the ERdj5 binding to the J-chain oligomers are necessary.
Owing to the practical difﬁculties in these measurements, it ishard to precisely estimate how many disulﬁde bonds are
reduced during one attack of an ERdj5 molecule against the
J-chain oligomers. Nevertheless, our biochemical experiments
showed that nearly 60% of the J-chain molecules were converted to the monomer after 60 min of the reaction at a 10:1 M
ratio of J-chain to ERdj5 (Fig. 1H). Thus, the rate constant for
the ERj5-catalyzed generation of the J-chain monomer iscalculated to be 1 × 10−1 min−1, indicating a low efﬁciency of
the reaction. Moreover, the HS-AFM analysis demonstrated
that the release of small J-chain fragments from the J-chain
oligomers occurred through repeated attacks of ERdj5, suggesting a low probability of disulﬁde reduction by one collision
with ERdj5. These observations indicate the stochastic natureof the ERdj5-mediated J-chain reduction and disaggregation.
This study reconstituted the likely pathway along which
misfolded nonglycoproteins are disaggregated by cooperation
between ERdj5 and BiP in the ER. Accumulation of misfolded
proteins stimulates the ER stress response and initiates the
ERAD machinery. Indeed, both BiP and ERdj5 are stressinducible (29, 30), which likely leads to further accelerationof ERAD pathways in cells. BiP is highly abundant in the ER
and multiple BiP partners or activators are also present(31–33). It is therefore possible that misfolded protein aggregates are ﬁrst handled by BiP and physically loosened tofacilitate subsequent disulﬁde reduction and/or oligomer
disassembly to smaller fragments. Consistently, preincubation
with BiP greatly enhanced the ERdj5-mediated resolution of

10 J. Biol. Chem. (2023) 299(11) 105274

J-chain oligomers (Fig. 2, G and J). At the latter stages, BiP isbelieved to engage in substrate transfer from ERdj5 to a retrotranslocation channel upon ATP hydrolysis (9, 10). Meanwhile, nucleotide exchange factors such as SIL1 nucleotide
exchange factor (SIL1) or glucose regulatory protein 170 likely
promote substrate release from the ADP-bound form of BiP byfacilitating the exchange of nucleoside diphosphate with
nucleoside triphosphate (34–36). It will be interesting todetermine whether these nucleotide exchange factors further
accelerate the disulﬁde reduction cycle catalyzed by ERdj5 and
BiP.
In conclusion, we conﬁrmed the signiﬁcant contribution of
BiP in scavenging disulﬁde-linked protein oligomers in concert
with ERdj5. The combinatorial approaches employed in thiswork deepen our understanding of the molecular mechanisms
underlying the ERdj5-mediated ERAD pathway. Detailed analyses of the spatial distributions and dynamics of ERdj5, BiP,
and other ERAD components using advanced microscopic
imaging technologies will provide in-depth, functional views of
how ERdj5 and BiP handle disulﬁde-linked oligomers for their
efﬁcient elimination in the ER.

Experimental procedures
Plasmid construction
ERdj5 mutants with an altered CXXC motif and ERdj5/
S557P were constructed using a PrimeSTAR Mutagenesis
Basal Kit (Takara Bio) and Gibson Assembly Master Mix (NewEngland BioLabs) with appropriate sets of primers. The geneencoding human BiP was cloned into the PET15b vector
containing an N-terminal His6-tag. The gene encoding humanJ-chain was cloned into the pCAGGS vector containing an Nterminal PA tag (37).
Cell culture and transient transfection
HEK293T cells were cultured in Dulbecco’s modiﬁed Eagle’s
medium (Nacalai Tesque) supplemented with 10% fetal bovine
serum (Nichirei) and antibiotics (100 U/ml penicillin and100 μg/ml streptomycin; Nacalai), in humidiﬁed air containing
5% CO2 at 37  C. Transfection of plasmids encoding FLAGtagged ERdj5 derivatives or PA-tagged J-chain was performed using PEI (Sigma-Aldrich) according to the protocol
recommended by the manufacturer. Approximately 2 ×
106 cells were precultured in 15 cm dishes (Thermo Fisher
Scientiﬁc) containing Dulbecco’s modiﬁed Eagle’s medium
supplemented with 4% fetal bovine serum. At 24 h after
plating, 20 μg of plasmid and 100 μg of 1 mg/ml PEI weremixed with 2 ml Opti-MEM (Gibco Service) for transfection,
followed by culturing in humidiﬁed air containing 5% CO2 at
37  C for 48 h.
Protein expression and puriﬁcation
Puriﬁcation of recombinant ERdj5 was performed at 4  C.
Harvested cell pellets were suspended in lysis buffer (50 mM
Tris–HCl pH 7.5, 150 mM NaCl, 10% v/v glycerol, 0.05% (w/v)
Tween-20, protease inhibitor cocktail; Nacalai Tesque) using a
potter-type homogenizer. Next, 1% (w/v) n-dodecyl-β-D-

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
maltoside (Nacalai Tesque) was added to the cell lysate andsamples were rotated. The mixture was centrifuged and thesupernatant containing target protein was recovered andresuspended with anti-FLAG beads (Sigma-Aldrich) in buffer
containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, and 10%(v/v) glycerol. After target protein binding, beads were washedwith resuspension buffer, and protein was eluted with buffer
containing 0.2 mg/ml FLAG peptide (Sigma-Aldrich). Samples
at each stage were collected and analyzed by Western blottingwith an anti-FLAG M2 antibody (F1804, Sigma-Aldrich,
1:20,000) to conﬁrm the presence of FLAG-tagged ERdj5. All
eluted fractions were mixed with 1-(3-sulfopropyl) pyridiniumhydroxide inner salt (NDSB201; Tokyo Chemical Industry) toa ﬁnal concentration of 1 M. Proteins were further puriﬁed bysize-exclusion chromatography with a Superdex200 10/300 GL
column (GE Healthcare) pre-equilibrated with buffer
composed of 20 mM Hepes pH 7.5, 10% (w/v) glycerol, 0.05%(w/v) Tween-20, and 1 M NDSB-201. Eluted fractions containing ERdj5 were collected, concentrated, and storedat −80  C. The ﬁnal yield of ERdj5 WT from 4 g of cells was
0.53 mg.
The puriﬁcation process for ERdj5 mutants with altered
CXXC motifs, H63A, and S557P, was the same as for WT
protein. The ﬁnal yield of each ERdj5 mutant was as follows:
0.1 mg for ERdj5 with altered CXXC motifs from 1 g of cells;
0.3 mg for ERdj5 H63A from 4 g of cells; and 0.04 mg for
ERdj5 S557P from 0.8 g of cells.
Puriﬁcation of recombinant BiP was performed using
Escherichia coli Rosetta (DE3) cells (Nippon Gene) carrying
expression plasmids for BiP. The cells were cultured at 37  Cuntil the OD600 reached 0.6, at which point expression ofrecombinant protein was induced by adding 0.5 mM IPTG
(Nacalai Tesque) and culturing was continued at 15  C for
18 h. The cytosolic extract was mixed with nickel-charged
afﬁnity resin (nickel-nitrilotriacetic acid [Ni-NTA], agarose;
QIAGEN) in Ni-NTA buffer (50 mM Tris–HCl pH 7.5,
500 mM NaCl, 2 mM MgCl2, 10% (v/v) glycerol, 0.2% Triton
X-100) and 20 mM imidazole (Nacalai Tesque). Ni-NTA
agarose beads that bound BiP were then washed with NiNTA buffer containing 3 mM ATP, and the protein was
eluted with Ni-NTA buffer containing 0.5 M imidazole. Flowthrough fractions containing BiP were further puriﬁed on a
MonoQ column (GE Healthcare). As the ﬁnal puriﬁcation
step, fractions containing target protein were applied to a
Superdex 200 10/300 GL column (GE Healthcare) equilibrated
in buffer containing 50 mM Tris–HCl pH 8, 150 mM NaCl,
10 mM MgCl2, and 10% (v/v) glycerol. Eluted fractions containing the target protein were collected and stored at −80  C,
and 0.4 mg of BiP was obtained from 2 l of culture.
Overexpression and puriﬁcation of PA-J-chain were performed with HEK293T cells transfected with a plasmid
overexpressing PA-J-chain using PEI and cultured for 48 h.
Harvested cells were lysed in lysis buffer (50 mM Hepes pH
7, 150 mM NaCl, 1% NP-40, 0.1% SDS, 10% (v/v) glycerol,
10 mM NEM, and protease inhibitor cocktail) and homogenized by sonication. Lysates were clariﬁed by centrifugationand precleared by incubation with Protein G Sepharose (GEHealthcare) for 1 h at 4  C with rotation. Protein G
Sepharose beads were removed by centrifugation and supernatants were incubated with anti-PA (NZ-27) antibody
beads (FUJIFILM Wako Pure Chemical Corporation) equilibrated with J-chain wash buffer (50 mM Hepes-NaOH pH
7.0, 150 mM NaCl, 10% (v/v) glycerol, 1% v/v Nonidet P-40,
0.1% w/v SDS) for 2 h at 4  C with gentle rotation. J-chain
oligomers were eluted by buffer containing 0.2 mg/ml PA
peptide (Sigma-Aldrich) and then further eluted with highconcentration magnesium solution. Samples at each stage
were collected and analyzed by Western blotting with ananti-PA antibody (015-25951, FUJIFILM Wako, 1:20,000) to
conﬁrm the presence of PA-J-chain. Eluates containing PAJ-chain were desalted by chromatography on a PD-10 column (GE Healthcare) and concentrated by a membrane ﬁlter(Amicon Ultra; Merck Millipore) with a 100 kDa cut-off. A
total of 0.23 mg PA-J-chain was obtained from 30 × 15 cm
dishes.
Concentrations of all puriﬁed proteins were measured by
the bicinchoninic acid method. All puriﬁed recombinant
proteins were stored at −80  C.
ATPase activity measurement
ATPase activity of BiP was analyzed by quantifying theamount of phosphate group (Pi) released during hydrolysis of
ATP by BiP. Quantiﬁcation of free Pi was performed using an
EnzChek Phosphate Assay Kit (Life Technologies corporation).
Recombinant BiP or ERdj5 were incubated in assay buffer
containing 20 mM Tris–HCl pH 7.5, 50 mM KCl, and 1.5 mM
MgCl2. Protein at 2 μM was used for each reaction. The reaction was initiated by addition of 1 mM ATP at 37  C. At
various timepoints, 30 μl aliquots were removed and boiled at
100  C for 5 min and then reacted with 2-amino-6-mercapto-7methylpurine riboside and purine nucleoside phosphorylase for
30 min at 22  C. In the presence of inorganic phosphate,
2-amino-6-mercapto-7-methylpurine riboside is converted
enzymatically by purine nucleoside phosphorylase to ribose
1-phosphate and 2-amino-6-mercapto-7-methylpurine, resulting in a spectrophotometric shift of the absorbance peak from
330 nm to 360 nm. The increase in absorbance at 360 nm wasmonitored using a U-3310 spectrophotometer (Hitachi). The
amount of free phosphate in the reaction solution was calculated from the increased absorbance, based on which theATPase activity of BiP was estimated.
J-chain oligomer reduction assay
Solutions containing proteins of interest, 2.5 mM GSH, and
1 mM ATP were prewarmed at 30  C for 10 min and mixed
well in assay buffer (50 mM Hepes pH 7, 0.05% (w/v) Tween20, 10% (v/v) glycerol, 1 mM MgCl2, and 1 M NDSB-201). The
reaction was started by mixing reagents with 5 μM J-chain
oligomers and stopped by addition of 10 mM NEM on ice at
different timepoints. Samples were separated by non-reducing
SDS-PAGE and detected by CBB staining. Band intensities
were quantiﬁed using Image Lab software (Bio-Rad Image Lab
Software | Bio-Rad).
J. Biol. Chem. (2023) 299(11) 105274

11

Disulﬁde bond reduction of ERAD substrates by ERdj5 and BiP
High-speed AFM observation
Single-molecule images of ERdj5 and J-chain oligomers
were acquired using a laboratory-built HS-AFM instrument(38, 39). Cantilevers (Olympus) were 6 to 7 μm long, 2 μm
wide, and 90 nm thick. The spring constant was 0.1 Nm−1,
and the resonant frequency and quality factor in water were
0.8 to 1 MHz and 2, respectively. The probe tip was grown
on a cantilever by electron beam deposition and further
sharpened by argon plasma etching (40). For AFM imaging,
the free oscillation amplitude was 1.2 nm, and the set-point
amplitude was 90% of the free oscillation amplitude. The
estimated tapping force was <30 pN (41). For observation, adroplet (2 μl) containing 100 nM J-chain oligomer diluted inbuffer containing 50 mM Tris–HCl pH 7, 150 mM NaCl,
5 mM MgCl2, and 2.5 mM GSH was loaded onto a cobaltscattered mica surface. After 3 min of incubation, the surface
was washed with buffer containing 50 mM Tris–HCl pH 7.0,
150 mM NaCl, 5 mM MgCl2, and 2.5 mM GSH to remove
unbound J-chain molecules. For the HS-AFM measurement ofJ-chain alone, the J-chain–immobilized mica surface wasincubated in the above buffer for 80 min before the acquisitionof the HS-AFM images. For the measurement of the snapshotsof the ERdj5-mediated J-chain reduction, ERdj5 (10 nM)
diluted in the same buffer was added to the cantilever holder
where the J-chain oligomer had been immobilized onto themica surface (no preincubation with GSH), and the acquisitionof the HS-AFM images was started immediately. Surface
coating with Co2+ prevented the adherence of excess ERdj5
molecules. All AFM observations were performed at room
temperature. Scan areas of 80 × 80 nm2 or 100 × 100 nm2 anda scan rate of 0.1 s/frame were used for standard observations.
HS-AFM images were analyzed using the laboratory-built
software Kodec4.4.7.39 (https://elifesciences.org/download/
aHR0cHM6Ly9jZG4uZWxpZmVzY2llbmNlcy5vcmcvYXJ0aW
NsZXMvMDQ4MDYvZWxpZmUtMDQ4MDYtY29kZTEtdjE
uemlw/elife-04806-code1-v1.zip?_hash=NtPTIc%2Bx0E4UJPn
ezJJDPvvASgWDc8D8Xl8I5oRoEik%3D) (24). A low-pass ﬁlter was applied to images to remove noise (24, 38). For identiﬁcation of observed particles, their areas and circularities
were calculated. Circularity is a measure of how circular theoutline of an observed molecule is deﬁned by the equation
4πS/L2, where L and S are the contour length of the outline
and the area surrounded by the outline, respectively. Thus, acircularity of 1 indicates a perfect circle, while values <1indicate a more extended conformation.

Data availability
All data described are contained within the article.
Supporting information—This
information.

articlecontains

supporting

Acknowledgments—We are grateful to Dr Hiroshi Kadokura for thetechnical instruction of culturing cells. We also thank Dr Zhenghao
Chen for the guidance and assistance in plasmid constructions andprotein puriﬁcations.

12 J. Biol. Chem. (2023) 299(11) 105274

Author contributions—X. C., S. I., and K. N. investigation; S. I.
project administration; K. N. and K. I. supervision; X. C. and K. I.
visualization; X. C. and K. I. writing-original draft; X. C., S. I., K. N.,
M. I., R. U., Y. K., K. N., and K. I. writing–review and editing.
Funding and additional information—This work was supported byfunding from AMED-CREST (21gm1410006h0001) to K. I., JSPS
KAKENHI to K. I. (18H03978, 21H04758, and 21H05247), and JST,the establishment of university fellowships toward the creation ofscience technology innovation to X. C. (JPMJFS2102).
Conﬂict of interest—The authors declare that they have no conﬂicts
of interest with the contents of this article.
Abbreviations—The abbreviations used are: BiP, immunoglobulinbinding protein; CBB, Coomassie brilliant blue; ER, endoplasmic
reticulum; ERAD, endoplasmic reticulum–associated degradation;
HPD, His-Pro-Asp; HS-AFM, high-speed atomic force microscopy;
NEM, N-ethylmaleimide; Trx, thioredoxin.

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