edem3, a soluble edem homolog, enhances glycoprotein erad
Post on 14-Feb-2017
223 Views
Preview:
TRANSCRIPT
1
EDEM3, a Soluble EDEM Homolog, Enhances Glycoprotein
ERAD and Mannose Trimming
Kazuyoshi Hirao*, †, ll, Yuko Natsuka
*, †, ll, Taku Tamura
‡, †, IkuoWada
‡, †, Daisuke
Morito*, †, Shunji Natsuka
§, †, Pedro Romero
¶, Barry Sleno
¶, Linda O. Tremblay
¶,
Annette Herscovics¶, Kazuhiro Nagata
*, †, Nobuko Hosokawa
*, †
From the *Department of Molecular and Cellular Biology, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto 606-8397, Japan, †CREST, JST, Japan, ‡Department ofCell Sciences, Institute of Biomedical Sciences, Fukushima Medical University School ofMedicine, Fukushima 960-1295, Japan, §Department of Chemistry, Osaka UniversityGraduate School of Science, Toyonaka 560-0043, Japan and ¶McGill Cancer Centre,
Montréal, Québec, Canada H3G 1Y6llThese two authors contributed equally to this work
Running title: EDEM homolog enhances glycoprotein ERAD
Address correspondence to: Nobuko Hosokawa, Department of Molecular and Cellular Biology,
Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan. Tel:
+81-75-751-3849; Fax: +81-75-751-4646; E-mail: nobuko@frontier.kyoto-u.ac.jp
Quality control in the endoplasmic
reticulum ensures that only properly folded
proteins are retained in the cell through
mechanisms that recognize and discard
misfolded or unassembled proteins in a
process called ERAD for endoplasmic
reticulum-associated degradation. We
previously cloned EDEM, ER degradation
enhancing aa-mannosidase-like protein, andshowed that it accelerates ERAD of
misfolded glycoproteins. We now cloned
mouse EDEM3, a soluble homolog of
EDEM. EDEM3 consists of 931 amino
acids and has all the signature motifs of
Class I aa-mannosidases (glycosyl hydrolasefamily 47) in its N-terminal domain and a
protease-associated motif in its C-terminal
region. EDEM3 accelerates glycoprotein
ERAD in transfected HEK293 cells, as
shown by increased degradation of
misfolded aa1-antitrypsin variant (null
Hong Kong, NHK) and of TCRaa.Overexpression of EDEM3 also greatly
stimulates mannose trimming not only
from misfolded NHK but also from total
glycoproteins, in contrast to EDEM which
has no apparent aa1,2-mannosidase activity.Furthermore, overexpression of the E147Q
EDEM3 mutant, which has the mutation in
one of the conserved acidic residues
essential for enzyme activity of
aa1,2-mannosidases, abolishes the
stimulation of mannose trimming and
greatly decreases the stimulation of ERAD
by EDEM3. These results show that
EDEM3 has aa1,2-mannosidase activity invivo suggesting that the mechanism
whereby EDEM3 accelerates glycoprotein
ERAD is different from that of EDEM.
http://www.jbc.org/cgi/doi/10.1074/jbc.M512191200The latest version is at JBC Papers in Press. Published on January 23, 2006 as Manuscript M512191200
Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
2
ER quality control is an elaborate
mechanism conserved from yeast to mammals
ensuring that newly synthesized proteins in the
ER fold and assemble correctly, and that only
proteins that acquire their correct
conformations are sorted further into the
secretory pathway (1-4). During this process,
proteins that fail to attain their native
conformation due to mutations of the
polypeptides or to ER stress conditions
adverse for protein folding, as well as orphan
subunits are degraded in a process known as
ERAD for ER-associated degradation (3, 5-7).
The recognition of misfolded proteins for
ERAD is still poorly understood, but there is
increasing evidence for a role of mannose
trimming in the targeting of glycoproteins for
ERAD (8, 9). In mammalian cells,
overexpression of ER a-mannosidase I
stimulates ERAD of misfolded glycoproteins
(10, 11) while the a1,2-mannosidaseinhibitors kifunensine and
1-deoxymannojirimycin stabilize misfolded
glycoproteins (12-16). These observations
suggested that Man8GlcNAc2 isomer B, the
major product of the ER a1,2-mannosidase, isa recognition marker for ERAD of
glycoproteins, but this view is being
challenged since there is increasing evidence
that trimming to smaller oligosaccharides
occurs on ERAD substrates (10, 17-19). We
previously cloned mouse EDEM (ER
degradation enhancing a-mannosidase-likeprotein) as a cDNA whose expression is
upregulated by ER stress, and showed that
EDEM accelerates glycoprotein ERAD (20).
EDEM is an integral ER membrane protein
that has all the signature motifs of Class I
a1,2-mannosidases (glycosyl hydrolase family47), but no detectable enzyme activity as a
processing a-mannosidase in vivo or in vitro.
Recently, it was found that EDEM extracts
terminally misfolded glycoproteins from the
calnexin cycle (21, 22). In S. cerevisiae, theER a1,2-mannosidase as well as
Htm1p/Mnl1p belonging to the same protein
family are also involved in ERAD since
disruption of the genes delays the ERAD of
glycoproteins (23, 24). Although EDEM and
Htm1p/Mnl1p were postulated to be lectins
involved in targeting misfolded glycoproteins
for ERAD, the precise mechanisms whereby
EDEM and Htm1p/Mnl1p recognize and sort
misfolded glycoproteins for degradation is still
unclear, and their role as lectins has not been
established directly. While this manuscript
was in preparation EDEM2 was reported to
stimulate ERAD of misfolded glycoproteins
without affecting mannose trimming (25, 26).
Here, we show that EDEM3 is a soluble
EDEM homolog located in the ER of
transfected mammalian cells that accelerates
ERAD of misfolded glycoproteins through a
mechanism likely to be different from that of
EDEM or EDEM2 since EDEM3 greatly
stimulates mannose trimming in vivo.
EXPERIMENTAL PROCEDURES
Cloning of mouse EDEM homolog—Five ESTclones were sequenced, and one clone
(G431003D06) which was kindly provided by
Dr. Y. Hayashizaki (RIKEN, Japan) (27)
contained the entire EDEM3 cDNA.
Sequencing was performed by PCR-based
dideoxy-termination method using BigDye v.
3 (ABI) and PCR 9700 (Perkin-Elmer), and
then analyzed with the ABI 3100 capillary
sequencer (ABI).
Plasmid construction—The coding region ofEDEM3 cDNA was subcloned into
pcDNA3.1+ by PCR, and the HA-tag was
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
3
introduced prior to the -KDEL ER retention
signal (EDEM3-HA). The NHK-QQQ mutant
was created by replacing Asn residues of
NHK glycosylation sites with Gln using the
Quick ChangeTMsite-directed mutagenesis kit
(STRATAGENE). The EDEM3 E147Q
mutant was constructed by the same method.
FLAG-tagged TCRa was kindly provided byDr. F. Tokunaga (Osaka City Univ. Grad. Sch.
Med.)
In vitro translation andtranslocation—EDEM3-HA cDNA was
linearized with EcoRI, and transcribed in vitroby T7 RNA polymerase (Promega). The
transcript was then translated in vitro in thereticulocyte lysate (Promega, Flexi-lysate)
supplemented with [35S]-methionine with and
without canine pancreas microsomes. The
localization of the EDEM3 product was
determined by the alkali-floatation method
(28), monitored by 10%SDS-PAGE. Mouse
EDEM tagged with HA was used as a control
ER membrane protein (20), and HSP47 as a
control ER lumenal protein (29).
Cell culture and transfection—HEK293,BALB/c3T3, COS7, and HeLa S3 cells
(provided by Japan Health Science Research
Resources Bank) were cultured in DMEM
supplemented with 10% fetal bovine serum
and antibiotics (100 U/ml penicillin G and 100
ng/ml streptomycin), in humidified air
containing 5% CO2 at 37oC. PC12h cells were
cultured in DMEM supplemented with 5%
fetal bovine serum and 5% horse serum.
HepG2 cells stably overexpressing the
EDEM3 were established using the pCX4bsr
plasmid which has a retrovirus LTR (30), and
selected in the presence of 30 mg/ml ofblasticidin (Funakoshi, Japan). Plasmids were
purified with a Plasmid Maxi Kit (Qiagen) and
transfected using the FuGene6 transfection
reagent (Roche) as described previously (10).
Northern blotting—Total RNA from culturedcells was extracted using TRIzol
TMreagent
(Invitrogen). After separation on
formaldehyde-denatured gel, RNAs were
blotted onto a nylon membrane (GeneScreen
plus, DuPont-NEN), and then were hybridized
with [32P]-labeled probes for 2 to 16 h in
PerfectHybTM
(TOYOBO, Japan)
hybridization solution. An MTNTM
Blot
membrane (Clontech) was used to examine
mouse tissue distribution. Probes for EDEM,BiP, b-actin, and HSP70 were labeled by themulti-primed labeling method
(Boehlinger-Mannheim). To detect EDEM3mRNA, probes were labeled by unidirectional
PCR using the 571 bp fragment of the EDEM3cDNA near its translational termination site, to
increase the specific activity of the probe, and
to avoid cross reactivity with EDEM.
Metabolic labeling, immunoprecipitation, andSDS-PAGE—Metabolic labeling, cell lysis,immunoprecipitation, and SDS-PAGE were
carried out as previously described (10),
except that DMEM lacking both methionine
and cysteine was used instead of DMEM
lacking methionine.
Antibodies—Antibodies against a1-AT werepurchased from DAKO (rabbit polyclonal).
Polyclonal antibodies against the HA-tag were
obtained from Santa Cruz Biotechnology,
anti-FLAG M2 antibody (mouse monoclonal)
was from Stratagene, and mouse monoclonal
antibody against PDI was purchased from
StressGen.
Reagents—Lactacystin was purchased fromKyowa Medics (Japan), and kifunensine was a
generous gift from Fujisawa Pharmaceutical
Co. (Osaka, Japan). EndoH and PNGaseF
(N-glycanaseF) were purchased from Roche
Applied Science or NEB.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
4
Immunocytochemistry—COS7 cells were
plated on a cover glass placed in a
3.5-cm-diameter dish approximately 24 h prior
to transfection. Twenty-four hours after
transfection, the cells were fixed with 4%
paraformaldehyde for 15 min at room
temperature, and incubated with anti-a1antibodies (rabbit polyclonal) and anti-PDI
antibody (mouse monoclonal) for 1 h, and
then with an Alexa546-labeled anti-rabbit and
an Alexa488-labeled anti-mouse antibody for
1h. Samples were examined by confocal
microscopy (LSM 510 META, Carl-Zeiss,
Germany).
Oligosaccharide analysis of NHK and totalcellular glycoproteins—Oligosaccharideanalysis of [
3H]-mannose-labeled NHK by
HPLC was performed as described previously
(10, 31). For oligosaccharide analysis of total
glycoproteins in HepG2 and cell line stably
overexpressing EDEM3, N-glycans were
released by hydrazinolysis, fluorescently
labeled using 2-aminopyridine, and were
fractionated by size on an NH2-P HPLC
column (Shodex Asahipak NH2P50, 0.46 x 15
cm, Showa Denko, Japan) as described
previously (32). Briefly, samples were loaded
onto the column and the oligosaccharides were
eluted at 0.5 ml/min by a linear gradient of
solvent A (acetonitrile) and solvent B (50 mM
ammonium acetate, pH 7.0) from 4:1 (v/v) to
3:7 (v/v) for 50 min. Oligosaccharides eluted
in each peak were identified by reverse phase
HPLC.
RESULTS
Cloning of EDEM3, a soluble homolog ofEDEM--A transcript named C1orf22 was
recently mapped in a region susceptible to
human hereditary prostate cancer (33). The
predicted ORF of C1orf22 has a sequence
similar to Class I a1,2-mannosidases (glycosylhydrolase family 47) and shows highest
homology to the KIAA0212 gene product (34).
Since KIAA0212 is the human ortholog of the
mouse EDEM gene (20), we searched the
mouse EST data base for the C1orf22
homolog. Sequencing of EST clones revealed
a mouse cDNA of 6,349 base
(GenBank/EMBL/DDBJ Accession number
AB188342), encoding a protein of 931 amino
acids (Fig. 1A). This ORF consists of a region
(amino acids 60-499) similar to Class I
a1,2-mannosidases (glycosyl hydrolase family47) followed by a C-terminal region
containing a protease-associated motif (amino
acids 686-780) (35) that is lacking in EDEM
and in all Class I a1,2-mannosidases studiedso far. It has a putative signal sequence at the
N-terminus as well as an ER-retrieval signal
(-KDEL) at its C-terminus.
The mouse EDEM3 cDNA sequence
exhibits 90% identity with its human ortholog
C1orf22 in the coding region, and 76% overall
identity. The mouse and human orthologs are
91% identical in amino acid sequence. The
mouse EDEM3 has 44 additional amino acids
at the N-terminus compared to the C1orf22
translation product. The a-mannosidasedomain shows 44% and 30% amino acid
identity between EDEM3 and EDEM, and
between EDEM3 and ER a-mannosidase I(ER ManI), respectively. All nine acidic
amino acids which are essential for a-1,2mannosidase activity (36) are conserved
between these three proteins (Fig. 1A),
although the two Cys important for activity of
the yeast ER a1,2-mannosidase (37) are notconserved in either EDEM3 or EDEM. The
PA motif is a consensus sequence found in
several proteases (35), the significance of
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
5
which in EDEM3 is currently unknown.
Expression of EDEM3 and effects of ERstress--Northern blotting shows a major
EDEM3 transcript of approximately 6.5 kb inall mouse tissues, as expected from the cloned
cDNA, with relatively high levels in liver,
heart and kidney (Fig. 2A), as reported for the
human homolog C1orf22 (33). EDEM mRNAis highly expressed in liver, moderately in
kidney, whereas the expression is low in heart,
brain and skeletal muscle (Fig. 2A). We
examined whether EDEM3 mRNA expressionis regulated by ER stress, as previously shown
for EDEM (20, 38). Addition of tunicamycin,
which induces ER stress by inhibiting
N-glycosylation of proteins, causes a mild
induction of EDEM3mRNA depending on thecell lines (Fig. 2B). The EDEM3 transcript isincreased about 1.5- 2-fold in BALB/c 3T3,
293 and PC12h cells, whereas no induction is
observed in COS7 and in HeLa S3 cells.
EDEM3mRNA is also upregulated by treatingcells with the glucose analogue
(2-deoxyglucose) or the calcium ionophore
A23187 (ER stress), but the level of EDEM3mRNA does not change in cells exposed to
cytosolic stress that greatly stimulates HSP70
expression (Fig. 2C).
EDEM3 is localized in the ER lumen--Toestablish whether the hydrophobic region near
the N-terminus acts as a cleavable signal
sequence upon co-translational translocation
into the ER, or whether it serves as a
transmembrane region, we separated integral
membrane proteins from soluble proteins by
alkali-floatation. When EDEM3 RNA is
translated in vitro, most of the radioactiveEDEM3 is recovered in the soluble fraction
(Fig. 3A). We observed a shift in the size by
SDS-PAGE of 110 kDa to 120 kDa when
EDEM3 was translocated into microsomes
(data not shown). Treatment with EndoH or
PNGaseF shows the removal of high-mannose
type N-glycans from EDEM3 (Fig. 3B), as
predicted from the sequence (Fig. 1B).
Next, we examined the intracellular
localization of EDEM3 by transfecting
HA-tagged EDEM3 transiently into COS7
cells. Indirect immunofluorescence shows a
fine reticular network pattern around the
nucleus that colocalizes with the ER resident
protein PDI (Fig. 3C). EDEM3 is not secreted
into the medium in pulse-chase experiments of
transiently transfected HEK293 cells (data not
shown). Thus, we conclude that EDEM3 is an
ER lumenal protein.
EDEM3 accelerates glycoprotein ERAD--Wethen investigated whether EDEM3 affects
glycoprotein ERAD. We used the
a1-antitrypsin genetic variant null (Hong
Kong) (NHK) as a soluble ERAD substrate
(13, 39). Co-transfection of EDEM3 enhances
the ERAD of NHK (Fig. 4A, B).
Co-immunoprecipitation of EDEM3 with
NHK was observed using antibodies to either
a1-AT or HA-tag (Fig. 4A), indicating thatEDEM3 interacts with NHK in the cells.
Co-immunoprecipitation was most prominent
after 1h and 2h chase. At these times the shift
in mobility of NHK indicates additional
trimming of the oligosaccharides. This
observation suggests that the interaction
between EDEM3 and NHK is stronger with
increased mannose trimming from NHK (Fig.
4A, compare lanes 5 and 6 with lane 4, and
lanes 8 and 9 with lane 7).
NHK degradation is inhibited by lactacystin
in the presence of co-transfected EDEM3,
showing that EDEM3 accelerates glycoprotein
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
6
ERAD by proteasomes (Fig. 4C). The
mobility shift of NHK after chase periods was
consistently larger in cells co-transfected with
EDEM3 than in cells co-transfected with
mock vector (Fig. 4A, compare lanes 4-6 with
1-3). The electrophoretic mobility shift of
NHK in EDEM3 overexpressing cells is
compared with that of deglycosylated NHK
prepared by PNGaseF digestion (Fig. 4D).
Addition of the a1,2-mannosidase inhibitorkifunensine greatly inhibits NHK degradation
in cells overexpressing EDEM3, and reduces
the mobility of NHK on SDS-PAGE (Fig. 4E).
This suggests that the mobility shift of NHK in
cells co-transfected with EDEM3 is caused by
the mannose trimming from the N-linked
oligosaccharides.
Since different mechanisms for ERAD of
soluble and transmembrane proteins have been
proposed (40-42), we examined the effect of
EDEM3 on a FLAG-tagged TCRa, a
glycosylated transmembrane ERAD substrate
(43). Co-expression of EDEM3 enhances the
degradation of TCRa-FLAG (Fig. 5A, B),
which is partly inhibited by kifunensine
treatment (Fig. 5A). However, EDEM3 does
not affect the degradation of NHK lacking all
three N-glycosylation sites (NHK-QQQ),
demonstrating its specificity for glycoproteins
(Fig. 5C, D). NHK-QQQ is degraded faster
than NHK which bears the three N-glycans,
and we have confirmed that NHK-QQQ was
also degraded by ERAD (data not shown).
These data indicate that the acceleration of
glycoprotein ERAD by EDEM3 depends on
mannose trimming from the N-glycans.
Effect of EDEM3 on mannose trimming fromN glycans of NHK and of total glycoproteins--Since the results in Fig. 4 suggested that
overexpression of EDEM3 stimulates
mannose trimming from N-glycans on NHK,
the oligosaccharides were examined after
labeling 293 cells with [3H]-mannose. The
N-glycans released from NHK by EndoH
were analyzed by HPLC. Overexpression of
EDEM3 greatly stimulates trimming of
N-glycans from NHK to Man7GlcNAc2 and
Man6GlcNAc2 (Fig. 6A, B), but there is
relatively little Man8GlcNAc2 found on NHK
in cells overexpressing EDEM3 compared to
mock transfected cells. At 0h chase, there is
only a trace amount of Man6GlcNAc2 and
Man7GlcNAc2 on NHK from mock
transfected cells whereas significant labeled
Man6GlcNAc2 and Man7GlcNAc2 are
observed in cells transfected with EDEM3.
The relative amount of Man6GlcNAc2 and
Man7GlcNAc2 increases with time of chase in
EDEM3 transfected cells and is greater at 1h
chase than the proportion of Man6GlcNAc2
and Man7GlcNAc2 in mock transfected cells.
Furthermore, at all time points the percent
labeled Man6GlcNAc2 and Man7GlcNAc2 is
much greater in EDEM3 transfected cells than
in the control. In contrast, the percent
radioactivity in Man8GlcNAc2 is always
much lower in the presence of EDEM3. This
pattern of oligosaccharides indicates that
overexpression of EDEM3 stimulates
mannose trimming from NHK. To determine
whether the increased mannose trimming is
due to intrinsic mannosidase activity of
EDEM3, the effects of the E147Q mutant on
NHK oligosaccharides was studied. Glu147is a
conserved residue corresponds to Glu132and to
Glu330in the active site of yeast and human ER
a1,2-mannosidases, respectively (44, 45). It isessential for enzyme activity since mutation of
this residue abolishes a1,2-mannosidaseactivity (36).
The pattern of oligosaccharides on NHK
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
7
obtained from cells overexpressing E147Q is
identical to that of mock transfected cells. And
the relative amounts of Man6GlcNAc2 and
Man7GlcNAc2 are the same as in control cells
(Fig. 6A, B).
Stimulation of mannose trimming by
EDEM3 was also demonstrated by analyzing
N-glycans of total glycoproteins from HepG2
cells stably overexpressing EDEM3 (Fig. 6C).
There is a large increase of Man6GlcNAc2
concomitant with a relative decrease of
Man7-8GlcNAc2, compared to the parental
HepG2 cells. All these results demonstrate that
EDEM3 has a1,2-mannosidase activity invivo.
Following labeling with
[35S]-methionine/cysteine, NHK degradation
was greatly reduced in 293 cells
overexpressing the E147Q mutant compared
to wild-type EDEM3 (Fig. 6D), indicating that
the mannosidase activity of EDEM3 is
important for its effect on ERAD of NHK.
DISCUSSION
The present work shows that EDEM3 is a
soluble homolog of EDEM that accelerates
ERAD of both soluble NHK and
membrane-bound TCRa in an
N-glycan-dependent manner. The extent of
ERAD stimulation on NHK degradation is
similar to that previously reported following
overexpression of EDEM and of ER ManI (10,
11, 20). However, the role of EDEM3 in
ERAD of NHK is likely to be different from
that of EDEM, since EDEM3 overexpression
greatly stimulates mannose trimming of
N-glycans from NHK whereas overexpression
of EDEM does not (10).
Furthermore, when EDEM3 is
overexpressed, the pattern of oligosaccharides
released from NHK is very different from that
observed on NHK isolated from ER
a1,2-mannosidase I transfected cells. In cellsoverexpressing EDEM3, there is extensive
trimming of N-glycans to Man6-7GlcNAc2
with relatively little Man8GlcNAc2 (Fig. 6A,
B) whereas in cells overexpressing ER ManI,
there is increased accumulation of
Man8GlcNAc2 and Glc1Man8GlcNAc2
concomitant with increased trimming to
smaller oligosaccharides (10). Importantly, the
effect of EDEM3 on the trimming of
N-glycans is abolished by mutating the
essential acidic residue Glu147to Gln (Fig. 6A,
B, E147Q), and the increased ERAD of NHK
due to EDEM3 overexpression is greatly
reduced by this mutation (Fig. 6D). This
residue is found in the active site of processing
Class I a-mannosidases by X-ray
crystallography (44-48) and is essential for
enzyme activity (36). Although these results
strongly indicate that EDEM3 stimulation of
NHK ERAD is caused by its
a1,2-mannosidase activity, an alternative lesslikely interpretation is that the mutation of
Glu147to Gln affects the conformation of
EDEM3 and thus abolishes the effect on NHK
degradation independently of enzyme activity.
Since overexpression of EDEM3 stimulates
mannose trimming from total glycoproteins as
well as from the misfolded glycoprotein NHK
(Fig. 6), EDEM3 is most likely acting as a
processing a1,2-mannosidase in vivo,accelerating trimming of Man8GlcNAc2
oligosaccharides to Man6-7GlcNAc2. Its
specificity appears to be different from that of
ER ManI that greatly stimulates trimming to
Man8GlcNAc2 and Glc1Man8GlcNAc2 (10).
The present results indicate that
Man8GlcNAc2 is not an exclusive targeting
signal for ERAD of glycoproteins and that
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
8
smaller oligosaccharides (Man5-7GlcNAc2)
attached to misfolded glycoproteins participate
in this recognition, in agreement with other
studies (10, 17-19, 49).
While this manuscript was in preparation,
two groups reported studies on another EDEM
homolog, which they named EDEM2 (25, 26).
Although EDEM2 stimulates glycoprotein
ERAD, it has no effect on mannose trimming
from misfolded glycoproteins, indicating that
its mechanism of action is different from that
of EDEM3 described in the present work. In
both manuscripts, the existence of EDEM3 is
mentioned, but its function is not further
analyzed.
The mechanisms involved in ERAD is an
area of active investigation at the present time,
not only for fundamental cell biology, but also
for clinical applications, because ERAD is
important in the pathogenesis of a large
number of genetic diseases caused by protein
misfolding. Yet the mechanisms whereby the
cell recognizes misfolded proteins and targets
them to ERAD are not fully understood. Since
earlier studies showed ER ManI and EDEM
both stimulate ERAD of glycoproteins, a
relatively simple mechanism has been
proposed whereby targeting of misfolded
glycoproteins depends on Man8B formed by
ERManI which is then recognized by EDEM.
However, it is clear from more recent studies
and from the work presented in this
manuscript that the targeting for ERAD is far
more complicated, since there are two
additional EDEM proteins implicated and
trimming of oligosaccharides on misfolded
glycoproteins to species smaller than Man8
occurs. Thus, the cloning and characterization
of EDEM3 makes a novel contribution to the
understanding of the quality control of
misfolded proteins.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
9
Abbreviations
ER: endoplasmic reticulum, ERAD: ER-associated degradation, EDEM: ER degradation enhancing
a-mannosidase-like protein, ER ManI: ER a1,2-mannosidase I, PA domain: protease-associateddomain, HA: influenza hemagglutinin epitope, a1AT: a1-antitrypsin, NHK: a1AT null (Hong Kong),TCRa: T cell receptor a subunit, Ab: antibody, BiP: Immunoglobulin heavy chain binding protein,PDI: protein disulfide isomerase.
Acknowledgements
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports and
Technology of Japan (to I.W., K.N and N.H.,), and by a grant from the Canadian Institutes of Health
Research (CIHR, to A.H.). We thank Dr. Y. Hayashizaki (RIKEN, Japan) for the cDNA clones, Dr. F.
Tokunaga (Osaka City Univ. Graduate Sch. Med) for the FLAG-tagged TCRa plasmid, and Ms. K.Kanamori for technical assistance.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
10
REFERENCES
1. Trombetta, E. S. & Parodi, A. J. (2003) Annu Rev Cell Dev Biol 19, 649-676.2. Ellgaard, L., Molinari, M. & Helenius, A. (1999) Science 286, 1882-1888.3. Fewell, S. W., Travers, K. J., Weissman, J. S. & Brodsky, J. L. (2001) Annu Rev
Genet 35, 149-191.4. Ellgaard, L. & Helenius, A. (2003) Nat Rev Mol Cell Biol 4, 181-191.5. Tsai, B., Ye, Y. & Rapoport, T. A. (2002) Nat Rev Mol Cell Biol 3, 246-55.6. Sitia, R. & Braakman, I. (2003) Nature 426, 891-894.7. McCracken, A. A. & Brodsky, J. L. (2003) Bioessays 25, 868-877.8. Cabral, C. M., Liu, Y. & Sifers, R. N. (2001) Trends Biochem Sci 26, 619-624.9. Helenius, A. & Aebi, M. (2004) Annu Rev Biochem 73, 1019-1049.10. Hosokawa, N., Tremblay, L. O., You, Z., Herscovics, A., Wada, I. & Nagata, K.
(2003) J Biol Chem 278, 26287-26294.11. Wu, Y., Swulius, M. T., Moremen, K. W. & Sifers, R. N. (2003) Proc Natl Acad Sci
U S A 100, 8229-8234.12. Liu, Y., Choudhury, P., Cabral, C. M. & Sifers, R. N. (1999) J Biol Chem 274,
5861-5867.13. Liu, Y., Choudhury, P., Cabral, C. M. & Sifers, R. N. (1997) J Biol Chem 272,
7946-7951.14. Chillaron, J., Adan, C. & Haas, I. G. (2000) Biol Chem 381, 1155-1164.15. Wilson, C. M., Farmery, M. R. & Bulleid, N. J. (2000) J Biol Chem 275,
21224-21232.16. Tokunaga, F., Brostrom, C., Koide, T. & Arvan, P. (2000) J Biol Chem 275,
40757-40764.17. Frenkel, Z., Gregory, W., Kornfeld, S. & Lederkremer, G. Z. (2003) J Biol Chem
278, 34119-34124.18. Kitzmuller, C., Caprini, A., Moore, S. E., Frenoy, J. P., Schwaiger, E., Kellermann,
O., Ivessa, N. E. & Ermonval, M. (2003) Biochem J 376, 687-696.19. Ermonval, M., Kitzmuller, C., Mir, A. M., Cacan, R. & Ivessa, N. E. (2001)
Glycobiology 11, 565-676.20. Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics,
A. & Nagata, K. (2001) EMBO Rep 2, 415-422.21. Molinari, M., Calanca, V., Galli, C., Lucca, P. & Paganetti, P. (2003) Science 299,
1397-1400.22. Oda, Y., Hosokawa, N., Wada, I. & Nagata, K. (2003) Science 299, 1394-1397.23. Jakob, C. A., Bodmer, D., Spirig, U., Battig, P., Marcil, A., Dignard, D., Bergeron, J.
J., Thomas, D. Y. & Aebi, M. (2001) EMBO Rep 2, 423-430.24. Nakatsukasa, K., Nishikawa, S., Hosokawa, N., Nagata, K. & Endo, T. (2001) J Biol
Chem 276, 8635-8638.25. Mast, S. W., Diekman, K., Karaveg, K., Davis, A., Sifers, R. N. &Moremen, K. W.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
11
(2005)Glycobiology 15, 421-436.26. Olivari, S., Galli, C., Alanen, H., Ruddock, L. &Molinari, M. (2005) J Biol Chem
280, 2424-2428.27. Carninci, P., Waki, K., Shiraki, T., Konno, H., Shibata, K., Itoh, M., Aizawa, K.,
Arakawa, T., Ishii, Y., Sasaki, D., Bono, H., Kondo, S., Sugahara, Y., Saito, R.,Osato, N., Fukuda, S., Sato, K., Watahiki, A., Hirozane-Kishikawa, T., Nakamura,M., Shibata, Y., Yasunishi, A., Kikuchi, N., Yoshiki, A., Kusakabe, M., Gustincich,S., Beisel, K., Pavan, W., Aidinis, V., Nakagawara, A., Held, W. A., Iwata, H., Kono,T., Nakauchi, H., Lyons, P., Wells, C., Hume, D. A., Fagiolini, M., Hensch, T. K.,Brinkmeier, M., Camper, S., Hirota, J., Mombaerts, P., Muramatsu, M., Okazaki, Y.,Kawai, J. & Hayashizaki, Y. (2003) Genome Res 13, 1273-1289.
28. Kutay, U., Ahnert-Hilger, G., Hartmann, E., Wiedenmann, B. & Rapoport, T. A.(1995) Embo J 14, 217-223.
29. Nagata, K. (1996) Trends Biochem Sci 21, 22-26.30. Akagi, T., Shishido, T., Murata, K. & Hanafusa, H. (2000) Proc Natl Acad Sci U S A
97, 7290-7295.31. Romero, P. A., Saunier, B. & Herscovics, A. (1985) Biochem J 226, 733-740.32. Hase, S. (1994)Methods Enzymol 230, 225-237.33. Sood, R., Bonner, T. I., Makalowska, I., Stephan, D. A., Robbins, C. M., Connors, T.
D., Morgenbesser, S. D., Su, K., Faruque, M. U., Pinkett, H., Graham, C., Baxevanis,A. D., Klinger, K. W., Landes, G. M., Trent, J. M. &Carpten, J. D. (2001)Genomics73, 211-222.
34. Nagase, T., Seki, N., Ishikawa, K., Ohira, M., Kawarabayasi, Y., Ohara, O., Tanaka,A., Kotani, H., Miyajima, N. & Nomura, N. (1996)DNA Res 3, 321-329, 341-354.
35. Luo, X. & Hofmann, K. (2001) Trends Biochem Sci 26, 147-148.36. Lipari, F. & Herscovics, A. (1999) Biochemistry 38, 1111-1118.37. Lipari, F. & Herscovics, A. (1996) J Biol Chem 271, 27615-27622.38. Yoshida, H., Matsui, T., Hosokawa, N., Kaufman, R. J., Nagata, K. &Mori, K.
(2003)Dev Cell 4, 265-271.39. Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H. & Woo, S. L. (1988) J
Biol Chem 263, 7330-7335.40. Caldwell, S. R., Hill, K. J. & Cooper, A. A. (2001) J Biol Chem 276, 23296-23303.41. Vashist, S., Kim, W., Belden, W. J., Spear, E. D., Barlowe, C. & Ng, D. T. (2001) J
Cell Biol 155, 355-368.42. Huyer, G., Piluek, W. F., Fansler, Z., Kreft, S. G., Hochstrasser, M., Brodsky, J. L. &
Michaelis, S. (2004) J Biol Chem 279, 38369-38278.43. Yu, H., Kaung, G., Kobayashi, S. & Kopito, R. R. (1997) J Biol Chem 272,
20800-20804.
44. Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W. & Howell, P. L. (2000) JBiol Chem 275, 41287-41298.
45. Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A. & Howell, P. L. (2000) Embo
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
12
J 19, 581-588.46. Lobsanov, Y. D., Vallee, F., Imberty, A., Yoshida, T., Yip, P., Herscovics, A. &
Howell, P. L. (2002) J Biol Chem 277, 5620-5630.47. Van Petegem, F., Contreras, H., Contreras, R. & Van Beeumen, J. (2001) J Mol Biol
312, 157-165.48. Tempel, W., Karaveg, K., Liu, Z. J., Rose, J., Wang, B. C. & Moremen, K. W.
(2004) J Biol Chem 279, 29774-29786.49. Movsichoff, F., Castro, O. A. & Parodi, A. J. (2005)Mol Biol Cell 16, 4714-4724.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
13
FIGURE LEGENDS
Figure 1. Similarity between EDEM3, EDEMand ERManI.
A. Amino acid sequence of mouse EDEM3. Region of similarity with Class I a1,2-mannosidasefamily (glycosyl hydrolase family 47), the protease associated domain, and the signal sequence are
shaded in orange, blue, and green, respectively. KDEL ER retrieval signal is underlined. The nine
conserved acidic amino acids are indicated by closed triangles, and putative N-glycosylation sites
are shown by dots.
B. Domain organization of mouse EDEM3, mouse EDEM and human ERManI.
Figure 2. Tissue distribution of EDEM3 and EDEM mRNAs and effect of stress on EDEM3mRNA in cultured cell lines.
A. Northern blots of mouse tissues showing EDEM3 and EDEM transcripts. Two mg of polyA RNAwere loaded (Clontech). Arrow indicates the position of EDEM3 mRNA of approx. 6.4 kb, andarrowheads show EDEMmRNAcorresponding to 5.8 and 2.4 kb.
B. Effect of tunicamycin treatment. Cells were treated with 5mg/ml tunicamycin (Tu) for 6-7 h.Twenty mg of total RNAwere analyzed by northern blotting with probes for EDEM3, EDEM, BiPand b-actin.
C. BALB/c 3T3 cells were subjected to ER stress (tunicamycin, 2-deoxyglucose, and A23187) or to
cytosolic stress (arsenite and heat shock). The same blot was rehybridized with BiP (induced byER stress), b -actin (loading control), and HSP70 (induced by cytosolic stress).
Figure 3. EDEM3 is expressed in the ER as a lumenal protein.
A. In vitro translation of [35S]-methionine-labeled EDEM3-HA. EDEM3-HA, Hsp47, and
EDEM-HA were translated in rabbit reticulocyte lysates supplemented with canine pancreas
microsomes. Proteins were separated into membrane and soluble fractions by alkali floatation,
and then analyzed by 10% SDS-PAGE and autoradiography. M: membrane fraction, S: soluble
fraction. The positions of the molecular weight markers are shown on the left side of the panels.
B. EndoH and PNGaseF digestion of EDEM3. 293 cells were transfected with EDEM3-HA, and
labeled for 3 h with [35S]-methionine/cysteine. After immunoprecitation by a-HA-tag Ab,
EDEM3-HA was digested with EndoH for 2.5 h, or with PNGaseF for 14 h at 37oC, and
separated by 10% SDS-PAGE. The positions of the molecular weight markers are indicated on
the left side of the panel.
C. Immunolocalization of EDEM3-HA. COS7 cells were transiently transfected with EDEM3-HA,
and stained with anti-HA Ab (2ndAb: Alexa546-labeled anti-rabbit) and anti-PDI Ab (2
ndAb:
Alexa488-labeled anti-mouse). Samples were examined by confocal microscopy. Bar indicates 10
mm.
Figure 4. EDEM3 accelerates glycoprotein ERAD.
A. Effect of EDEM3 on NHK degradation. 293 cells were transiently transfected with NHK and
EDEM3-HA/mock, pulse-labeled for 15 min with [35S]-methionine/cysteine, and then chased for
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
14
the periods indicated. After immunoprecipitation using anti-a1AT or anti-HA Ab, samples wereseparated by 10% SDS-PAGE until the Evans Blue reaches near the bottom of the gel. The
positions of the molecular weight markers are shown on the left side of the panel.
B. Quantification of NHK degradation. Average of three independent experiments were plotted with
standard deviations. Relative radioactivity of NHK at chase 0 h was set to 100%.
C. Effect of proteasome inhibition. 293 cells were transfected with NHK and EDEM3-HA, and
treated with (+) or without (-) lactacystin (20 mM) added 3 h prior to pulse-labeling with[35S]-methionine/cysteine for15 min, followed by chase for the times indicated. Lactacystin was
also present during the chase in lactacystin-treated samples. NHK immunoprecipitated with
a1-AT Ab was subjected to 10% SDS-PAGE. Arrow indicates the position of NHK after a 15min pulse.
D. Effect of PNGaseF on NHK. NHK expressed in 293 cells was immunoprecipitated as described in
C, and the electrophoretic mobility shift of NHK in EDEM3-transfected cells during chase was
compared with that of deglycosylated NHK prepared by PNGaseF digestion. The positions of the
molecular weight markers are indicated on the left side of the panel.
E. Effect of kifunensine on NHK degradation. Cells were metabolically labeled with
[35S]-methionine/cysteine for 15 min (P: pulse) and chased for 90 min (C: chase). In
kifunensine-treated cells, drug (5 mg/ml) was added 3 h prior to pulse-labeling and during thechase.
Figure 5. EDEM3 accelerates ERAD of membrane-bound TCRaa but not unglycosylated NHK.A. Effect of EDEM3 on TCRa degradation. 293 cells were transiently transfected with TCRa-FLAGand EDEM3-HA/mock, pulse-labeled for 15 min with [
35S]-methionine/cysteine, and then chased
for the periods indicated. After immunoprecipitation using anti-FLAG Ab, samples were separated
by 10% SDS-PAGE. Kifunensine (5 mg/ml) was added 3 h prior to pulse-labeling and during thechase where indicated (+). The positions of the molecular weight markers are shown on the left
side of the panel.
B. Quantification of TCRa degradation. Average of three independent experiments were plotted withstandard deviations. Relative radioactivity of TCRa at chase 0 h was set to 100%.
C. Effect of EDEM3 on the degradation of unglycosylated NHK-QQQ. 293 cells were transfected
with NHK-QQQ and EDEM3-HA/mock, and pulse-chase experiment was performed as described
in A. NHK-QQQ immunoprecipitated with a1-AT Ab was subjected to 10% SDS-PAGE.
Arrowhead indicates the position of NHK-QQQ. The position of the molecular weight marker is
shown on the left side of the panel.
D. Quantification of NHK-QQQ degradation. Average of three independent experiments were plotted
with standard deviations. Relative radioactivity of NHK-QQQ at chase 0 h was set to 100%.
Figure 6. EDEM3 stimulates mannose trimming of N-linked oligosaccharides.
A. Effect of EDEM3 on mannose trimming of N-linked glycans on NHK. 293 cells were transiently
transfected with NHK and either mock or EDEM3. After labeling for 30 min in medium
containing 1mM glucose and [2-3H]-mannose, cells were chased in medium containing 25mM
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
15
glucose with or without lactacystin (20 mM). Immunoprecipitated NHK was separated by
SDS-PAGE, and transferred to PVDF membrane. NHK bands were excised from the blotted
membrne, and the oligosaccharides were released with endo-H and fractionated by HPLC. Arrows
indicate the position of the [14C]-labeled Glc3Man9GlcNAc internal standard.
B. Effect of EDEM3 E147Q mutant on mannose trimming of N-linked glycans on NHK. 293 cells
were transfected with NHK and either mock, EDEM3 or E147Q mutant plasmids. Samples were
analyzed as described in A, and relative amount of each oligosaccharides are shown as bar graph.
C. Effect of EDEM3 overexpression on oligosaccharides from total cellular glycoproteins from
HepG2 cells. The fluorescent-labeled oligosaccharides were fractionated by HPLC as described in
materials and methods.
D. Effect of the EDEM3 E147Q mutant on NHK degradation in 293 cells. Pulse-chase experiment
using [35S]-methionine/cysteine was performed as described in Fig. 4A. The average of four
independent experiments is shown on the graph.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
MSKAGGCRGCGCRVPQRASWSLVAATAALCLVLATSVCTAGAAPMSREEKQKLGNQVLEMFDHAYGNYMEHAYPADELMP 80
LTCRGRVRGQEPSRGDVDDALGKFSLTLIDSLDTLVVLNKTKEFEDAVRKVLRDVNLDNDVVVSVFETNIRVLGGLLGGH 160
SLAIMLKEKGEHMQWYNDELLHMAKQLGYKLLPAFNTTSGLPYPRINLKFGIRKPEARTGTETDTCTACAGTLILEFAAL 240
SRFTGATIFEEYARKALDFLWEKRQRSSNLVGVTINIHTGDWVRKDSGVGAGIDSYYEYLLKAYVLLGDDSFLERFNTHY 320
DAIMRYISQPPLLLDVHIHKPMLNARTWMDALLAFFPGLQVLKGDIRPAIETHEMLYQVIKKHNFLPEAFTTDFRVHWAQ 400
HPLRPEFAESTYFLYKATGDPYYLEVGKTLIENLNKYARVPCGFAAMKDVRTGSHEDRMDSFFLAEMFKYLYLLFADKED 480
IIFDIEDYIFTTEAHLLPLWLSTTNRSISKKNTTSEYTELDDSNFDWTCPNTQILFPNDPLYAQSIREPLKNVVDKSCPR 560
GIIRVEESFRSGAKPPLRARDFMATNPEHLEILKKMGVSLIHLKDGRVQLVQHAIQAASSIDAEDGLRFMQEMIELSSQQ 640
QKEQQLPPRAVQIISHPFFGRVVLTAGPAQFGLDLSKHKETRGFVASSKPYNGCSELTNPEAVMGKIALIQRGQCMFAEK 720
ARNIQNAGAIGGIVIDDNEGSSSDTAPLFQMAGDGKDTDDIKIPMLFLFSKEGSIILDAIREHKQVEVLLSDKARDRDPE 800
MENEDQPSSENDSQNQSAEQMLSLSQTVDLADKESPEHPADSHSEASPSDSEEAAGFAPSEQISGSTENHETTSLDGECT 880
DLDNQVQEQSETEEDSSPNVSWGTKAQPIDSILADWNEDIEAFEMMEKDEL 931.
..
..
.
.A
B
glycosyl hydrolase family 47 domain
transmembrane region putative N-glycans
protease associated domain
ER ManI
EDEM
EDEM3ERretrievalsignal
signalsequence
Figure 1
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
9.5
4.4
7.5
1.35
2.4
kb
hear
tbr
ainsp
leen
lung
liver
skele
tal m
uscle
kidne
yte
stis
EDEMEDEM3
9.57.5
4.4
2.4
1.35
kb
hear
tbr
ainsp
leen
lung
liver
skele
tal m
uscle
kidne
yte
stis
A
B
BiP
EDEM3
CO
S7
HEK
293
HeL
aS3
- + - + - + - + - +Tu
PC12
h
BALB
/c3T
3
2.4k
5.8k
EDEM
β-actin
EDEM3
BiP
β-actin
HSP70
cont
rol
tuni
cam
ycin
A231
87ar
seni
tehe
atsh
ock
2-de
oxyg
luco
se
C
Figure 2 by guest on A
pril 12, 2018http://w
ww
.jbc.org/D
ownloaded from
PDIEDEM3-HA merge
B
M SEDEM-HA
82.6 kDa
M S
Hsp47M S
EDEM3-HA
130 kDa
A
C
- + - +
EDEM3-HA
EndoH PNGaseF
deglycosylatedEDEM3-HA
82.6 kDa
130 kDa
Figure 3
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
100
50
010
1 2
(%)
(h)Chase
mockEDEM3
Rel
ativ
era
dioa
ctiv
ity
NHK
EDEM3150
250
100
50
75
0 1 2 0 1 2 0 1 2 (h)Chase
EDEM3 - ++α-α1AT AbIP α-HA Ab
lane 1 2 3 4 5 6 7 8 9
0 45 90 0 45 90 (min)
- +
+EDEM3
Lactacystin
Chase
NHK
lane 1 2 3 4 5 6
NHK
P C P C P C P CPulse/Chase
Kifunensine
EDEM3
+ +- -- +
lane 1 2 3 4 5 6 7 8
C D
BA
Figure 4
E
0 1 2 -
+
+EDEM3
Lactacystin
Chase (h)
NHK
-
NHK-CHO
+ PNGaseF
-
50 kDa
37 kDa
lane 1 2 3 4 5
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
100
50
010
1 2
(%)
(h)Chase
mockEDEM3
0 1 2 0 1 2 (h)Chase
EDEM3 - +
NHK-QQQ40.8 kDa
lane 1 2 3 4 5 6
BA
Figure 5
0 45 90 0 45 90 0 45 90 0 45 90 (min)
KIF
plasmid mock
α-FLAG AbIP
EDEM3
- + - +chase
TCRα-FLAG
lane 1 2 3 4 5 6 7 8 9 10 11 12
82.6
130
200
31.5
40.8
100
50
01
1 2
(%)
(h)Chase
10
5
mockEDEM3
D
C
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
MOCKEDEM3E147Q
chase
0
10
20
0 1 2
M6
(h)
(%)
0
20
40
0 1 2
G1M9
0
20
40
0 1 2
M7
0
10
20
0 1 2
M8
0
35
70
0 1 2
M9
A
100
50
010
1 2
(%)
(h)Chase
EDEM3
E147Qmock
Rel
ativ
era
dioa
ctiv
ity
0 1 2 0 1 2 0 1 2 (h)Chase
plasmid mock
α-α1AT AbIP
EDEM3 E147Q
NHK
B
C
M9
G1M9M8M7
M6
M5
27.5 30.0 32.5
10
0
20
M9
G1M9
M8
M7
M6M5
27.5 30.0 32.5 35.0
40
0
80
Flu
ores
cenc
ein
tens
ity
Retention time (min)
EDEM3-HepG2
HepG2
D
EDEM3
E147Q
Mock
Rad
ioac
tivity
(dpm
)
Chase 0 h Chase 1 h Chase 2 h
Fraction Number
Figure 6 by guest on A
pril 12, 2018http://w
ww
.jbc.org/D
ownloaded from
Nagata and Nobuko HosokawaNatsuka, Pedro Romero, Barry Sleno, Linda O. Tremblay, Annette Herscovics, Kazuhiro
Kazuyoshi Hirao, Yuko Natsuka, Taku Tamura, Ikuo Wada, Daisuke Morito, Shunjitrimming
EDEM3, a soluble EDEM homolog, enhances glycoprotein ERAD and mannose
published online January 23, 2006J. Biol. Chem.
10.1074/jbc.M512191200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
top related