From the Centre for Biotechnology, Department of
Biosciences at Novum, Karolinska Institute, and the
** Steroid Group, Södertörns Högskola,
S-14157 Huddinge, Sweden, ¶ Department of Biological and
Technological Research, San Raffaele Scientific Institute, and
the §§ Università Vita-Salute San
Raffaele, 20132 Milan, Italy, and the
Department of Developmental Biology,
Tampere University Medical School, and the Department of Pathology,
Tampere University Hospital, Fin-33101 Tampere, Finland
Received for publication, July 12, 2002, and in revised form, October 25, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A complex array of chaperones and enzymes reside
in the endoplasmic reticulum (ER) to assist the folding and assembly of
and the disulfide bond formation in nascent secretory proteins. Here we
characterize a novel human putative ER co-chaperone (ERdj5) containing
domains resembling DnaJ, protein-disulfide isomerase, and thioredoxin
domains. Homologs of ERdj5 have been found in Caenorhabditis
elegans and Mus musculus. In vitro
experiments demonstrated that ERdj5 interacts via its DnaJ domain with
BiP in an ATP-dependent manner. ERdj5 is a ubiquitous
protein localized in the ER and is particularly abundant in secretory
cells. Its transcription is induced during ER stress, suggesting
potential roles for ERdj5 in protein folding and translocation across
the ER membrane.
The endoplasmic reticulum
(ER)1 is the site where
membrane and secretory proteins fold, assemble, and form disulfide
bonds. The ER provides a suitable environment for these processes, with optimal pH and redox conditions and a vast array of chaperones and
enzymes available, including protein-disulfide isomerase (PDI), BiP
(immunoglobulin heavy chain-binding protein),
GRP94, and calnexin (1-3). The ER exerts also a tight quality control
on its protein products; as a result, only proteins that attain their
native state are transported to the Golgi. Proteins that fail to fold or assemble are dislocated to the cytosol for proteasomal destruction (4, 5). In both yeast and mammalian cells, the accumulation of
misfolded polypeptides in the ER lumen results in the transcriptional up-regulation of many genes encoding ER-resident chaperones and folding
catalysts, a process known as the unfolded protein response (UPR) (6,
7). Genes induced during the UPR share regulatory sequences, ER stress
elements (ERSEs), or a UPR element (6, 8-10).
Like other members of the Hsp70 family, BiP interacts with newly
synthesized polypeptides in ATP-dependent cycles of binding and release that are controlled by co-chaperone molecules of the DnaJ
family. Because the intrinsic ATPase activity of Hsp70 proteins is weak (11), DnaJ molecules play a crucial role in catalyzing these
reactions, stimulating the ATPase activity, and facilitating substrate
binding (12, 13). DnaJ proteins can be divided into three subgroups
based upon their degree of conservation in relation to
Escherichia coli DnaJ. Type I DnaJ proteins contain an
N-terminal J domain, a G/F-rich region, and a cysteine-rich
Zn2+-binding region. Type II DnaJ proteins lack the
cysteine-rich domain, whereas type III proteins contain only the J
domain (14). The latter mediates the interaction with the Hsp70
partner(s) via a conserved His-Pro-Asp motif.
Catalysis of oxidative folding is necessary to rapidly generate the
correct disulfide bonds in newly synthesized proteins (15). A crucial
component of the ER folding machinery is PDI. This multifunctional
protein catalyzes disulfide bond formation and isomerization within the
ER and displays both chaperone and anti-chaperone activities, depending
on its redox state (15, 16). PDI needs to be continually reoxidized to
be effective as a catalyst for disulfide bond formation. Members of the
Ero1 family carry out this reoxidizing function in yeast and mammalian cells (17-19). The structure of PDI encompasses five domains: four consecutive domains (a-b-b'-a') followed by a
c region, which is a putative, low-affinity, high-capacity
Ca2+-binding site (15). The two CXXC
protein-thiol oxidoreductase active sites are located in the
a and a' domains, which display homologies to
thioredoxin (20). Despite their low sequence homology to the
a domain, the b and b' domains also
adopt a thioredoxin fold. Therefore, PDI is assumed to be composed of
redox active and inactive thioredoxin modules (20).
In addition to PDI, the ER contains numerous other proteins endowed
with oxidoreductase activity (ERp72, ERp57, p5, etc.). The abundance of
these proteins probably reflects the high demand for efficiently
forming, isomerizing, and also reducing disulfide bonds in proteins
that are undergoing folding and quality control in this organelle (21).
The interactions between ERp57 and either calnexin or calreticulin
underlie the necessity of coordinating folding with disulfide bond
formation. We report here the identification and
characterization of a novel ER-resident molecule (ERdj5) that features
a DnaJ domain, a PDI-like domain, and a thioredoxin domain, suggesting
a role as an ER folding assistant.
cDNA Cloning of Human ERdj5--
BLAST (22) was used to
identify human expressed sequence tag (EST) clones encoding proteins
similar to thioredoxin. Some of these EST entries were found to encode
a putative protein containing both a DnaJ domain and four putative
thioredoxin-like active sites. Based on these sequences, the following
primers were designed and used for 5'- and 3'-RACE/PCR in human testis
and adrenal gland cDNA libraries (Clontech) to
isolate human ERdj5 cDNA: human ERdj5-F1 and ERdj5-F2,
nucleotides 1960-1985 and 1344-1359, respectively (nucleotide numbers
refer to GenBankTM/EBI accession number AF038503); and
human ERdj5-R1 and ERdj5-R2, nucleotides 2479-2455 and 2518-2482,
respectively. The resulting sequences were used to amplify by PCR the
full-length cDNA of ERdj5 from the same libraries.
Glutathione S-Transferase (GST) Purification of the DnaJ Domain
of ERdj5 and Full-length ERdj5--
The DnaJ domain was amplified by
PCR using primers ERdj5/GST-F1 (nucleotides 438-468) and ERdj5/GST-R1
(nucleotides 672-701), containing BamHI and
EcoRI restriction sites, respectively. The product was
digested with BamHI and EcoRI and cloned into the pGEX-4T-1 vector. A fusion protein between GST and the DnaJ domain of
ERdj5 (GST-DnaJ) was purified from E. coli BL21(DE3) cells by growing a 250-ml culture to A600 ~ 0.6 and
then inducing protein expression by adding 1 mM
isopropyl-1-thio-
Full-length ERdj5 was amplified by PCR using the specific
forward and reverse primers ERdj5/GST-F1 (see above) and
ERdj5/GST-R2 (nucleotides 2727-2756), including BamHI and
SalI restriction sites, respectively; digested; and
cloned into the corresponding sites in the pGEX-4T-1 vector. GST-ERdj5
fusion protein was purified from E. coli BL21(DE3) cells
according to the previously described protocol (23). GST-ERdj5 fusion
protein was extensively dialyzed following purification.
Northern Blot Analysis--
Human multiple-tissue Northern blots
with poly(A)+ RNA from different tissues were purchased
from Clontech. The human ERdj5 open
reading frame (ORF) and the human PDI ORF were labeled with [ Green Fluorescent Protein (GFP) with ERdj5 Plasmid
Generation--
First, the pEGFP-N3 plasmid was modified so
that the ER retention signal would be at the end of the GFP sequence.
PCR was used with specially designed primers including the appropriate restriction sites, the stop codon, and the ER retention signal KDEL to
amplify GFP-KDEL. This product was ligated into the pGEMT-easy plasmid
and sequenced to confirm the presence of the KDEL sequence. The GFP DNA
was released from the pEGFP-N3 plasmid by digestion with the
restriction enzymes NotI and EcoRI. The modified
GFP-KDEL was released from the pGEMT-easy plasmid by digestion with
NotI and EcoRI and subsequently cloned into the
empty pEGFP-N3 NotI and EcoRI sites (also
sequenced to confirm KDEL presence). This modified plasmid (pEGFP-KDEL)
was used for GFP localization studies. The ERdj5 sequence
not including the ER retention signal sequence (KDEL) was amplified
using forward and reverse primers containing XhoI and
BamHI restriction sites, respectively, and the Kozak sequence (24). This ERdj5 product was digested and ligated
in-frame into pEGFP-KDEL.
Confocal Microscopy Localization Studies of GFP-tagged
ERdj5--
HEK293 cells were chosen for these studies, as they are
epithelial kidney adherent cells that are easily transiently
transfected. The 293 cell line is a permanent line of primary human
embryonic kidney cells transformed by sheared human adenovirus type 5 DNA (American Type Culture Collection, Manassas, VA). HEK293 cells were
grown in 1:1 Dulbecco's modified Eagle's medium/nutrient mixture F-12
supplemented with fetal calf serum and 10 µg/ml gentamycin and
transiently transfected using polyethyleneimine (Aldrich). 1 µg of
GFP- plus KDEL-tagged ERdj5 DNA and 1 µg of pDsRed2-ER (Clontech) DNA were diluted in 20 µl of
H2O, and 1 µl of 0.1 M polyethyleneimine was
added. The mixture was vortexed, incubated at room temperature for 10 min, and subsequently added to the medium and applied to the cells.
48 h after transfection, live GFP pictures were acquired with a
Leica laser scanning confocal microscope. GFP was excited at a
wavelength of 488 nm using an argon/krypton laser, and emitted light
was collected between 500 and 540 nm; and for excitation of
pDsRed2-ER (Clontech), we used the 568-nm
line, and emitted light was collected between 570 and 620 nm.
Generation and Purification of His-tagged BiP--
The
BiP cDNA including the signal peptide was amplified from
a human liver cDNA library by PCR using the specific forward and
reverse primers BiP-F5 (nucleotides 131-160) and BiP-R1 (nucleotides 2164-2180), corresponding to GenBankTM/EBI accession
number X87949 for Homo sapiens BiP
mRNA. The product was ligated into the pGEMT-easy vector. To
generate suitable restriction sites for cloning into the pET-15b
plasmid for subsequent protein purification, PCR was carried out using
primers BiP-pET-F (nucleotides 268-291) and BiP-pET-R (nucleotides
2167-2193), containing NdeI and BamHI
restriction sites, respectively, with pGEM-BiP cDNA as template.
Following digestion with BamHI and NdeI, the product was ligated in-frame into the His-tagged pET-15b plasmid (Novagen). His-tagged BiP was purified essentially following a previous
protocol (25).
Surface Plasmon Resonance Interaction Studies--
Surface
plasmon resonance was carried out using a BIAcore2000 instrument
(BIAcore, Uppsala, Sweden). Using amine coupling, anti-GST antibodies
(~6000 response units) were immobilized onto all surfaces of a
CM5 sensor chip (BIAcore). Subsequently, equal molar quantities of GST
and the GST-DnaJ and GST-ERdj5 fusion proteins were captured. All
proteins were diluted in HBS-EP (10 mM HEPES, pH
7.4, 150 mM NaCl, 3 mM EDTA, 0.005%
polysorbate) (BIAcore) supplemented with 5 mM
MgCl2 and injected at a flow rate of 5 µl/min. The GST
signal was used for background correction.
GST Pull-down Experiments with in Vitro Translated BiP and
His-tagged BiP--
The BiP ORF cloned into the pET-15b
vector from the previous BiP protein purification was used as template
for in vitro translation. Protein was synthesized in
vitro using the T7 RNA polymerase-based rabbit reticulocyte
lysate-coupled transcription/translation kit (TNT, Promega) and labeled
with [35S]Met. Briefly, GST, GST-DnaJ, and GST-ERdj5
(~2 µg each) bound to glutathione-Sepharose 4B beads were incubated
for 2 h upon rotating at 4 °C with 4 µl of in
vitro translated [35S]Met-labeled protein in
incubation buffer (50 mM potassium inorganic phosphate (pH
7.4), 100 mM NaCl, 1 mM MgCl2, 10%
glycerol, and 0.1% Tween) containing 1.5% bovine serum albumin. (As a
control, GST and the GST fusion proteins were incubated under the same conditions without in vitro translated BiP protein.) The
beads were washed three times for 15 min with incubation buffer without bovine serum albumin, resuspended in 100 µl of 2× SDS sample buffer, boiled for 5 min, and pelleted in a microcentrifuge. 15 µl of each supernatant was subjected to SDS-PAGE. To control the stability of
the GST fusion proteins and equal loading, gels were stained with
Coomassie Blue, incubated in Amplify liquid (Amersham Biosciences) for
15 min, and dried under vacuum before autoradiography.
GST pull-down assays using His-tagged BiP were carried out essentially
as described above. Briefly, 2 µg of purified His-tagged BiP protein
was added to ~2 µg of GST, GST-DnaJ, or GST-ERdj5 bound to
glutathione-Sepharose 4B beads in the presence of 2 mM ATP
or ADP and incubated overnight at 4 °C. Following SDS-PAGE, proteins
were transferred to Hybond-C super membrane (Amersham Biosciences) using standard Western blotting techniques. Monoclonal anti-His tag antibody (Clontech) was used at a
dilution of 1:2000 with horseradish peroxidase-linked sheep anti-mouse
Ig (Amersham Biosciences) used as the secondary antibody at a
dilution of 1:5000.
In Situ Hybridization of Mouse ERdj5--
For in situ
hybridization, adult male and female mice (2-3 months of age) were
killed with carbon dioxide, and the tissues were excised and frozen on
dry ice. The samples were sectioned with a Microm HM500 cryostat
at 14 µm and thaw-mounted onto polylysine glass slides (Menzel). The
sections were stored at ERdj5 mRNA Expression and UPR Induction--
HEK293T cells
(American Type Culture Collection), an easily transfectable derivative
of HEK293 cells expressing SV40 T antigen, were grown in Dulbecco's
modified Eagle's medium with 5% fetal calf serum. Total cellular RNA
preparation and Northern blot analysis were carried out as described
previously (27). To induce the UPR or unrelated stress
response, cells were cultured for 6 h in the presence of
dithiothreitol (2 mM), tunicamycin (10 µg/ml), thapsigargin (2 µM), A23187 (2 µM), EGTA (2 mM), or deoxyglucose (10 mM); deprived of fetal
calf serum; UV light-irradiated; or incubated at 42 °C for 30 min.
cDNA Cloning of ERdj5 and Sequence Analysis--
A screen of
the GenBankTM/EBI EST Data Bank led to the identification
of sequences containing putative thioredoxin active sites. 5'- and
3'-RACE/PCR analyses were performed using specially designed primers in
both human testis and adrenal gland cDNA libraries to clone the
full-length cDNA. The PCR product was cloned and sequenced,
confirming the sequence obtained from the ESTs. The complete sequence
reveals an ORF of 2379 base pairs, with 5'- and 3'-untranslated regions
of 415 and 1384 base pairs, respectively, surrounding an ORF that
encodes a putative protein of 793 amino acids with an estimated
molecular mass of 91 kDa and a pI of 7.03 (Fig.
1). The protein was named ERdj5 following
the nomenclature proposed for ER proteins containing a DnaJ domain in
the recent study by Shen et al. (28). After an N-terminal
hydrophobic sequence, a domain with the features of DnaJ/Hsp40
proteins, including the His-Pro-Asp motif, is encountered (Fig.
2A). Neither G/F-rich nor
cysteine-rich regions are present, thus making ERdj5 a type III DnaJ
protein (14, 29). Following the DnaJ domain are four thioredoxin-like
domains, with different putative redox active sites, each containing a
CXXC motif. The last domain has a typical thioredoxin
signature, WCGPC. A KDEL tetrapeptide is present at the C terminus,
possibly mediating ER retention (see below).
Genomic Organization and Chromosomal Localization of
ERdj5--
The gene encoding ERdj5 has been
identified in genomic data bases and mapped to human chromosome 2 at
positions p22.1-23.1. Several disease genes have been mapped to this
region, including a form of recessive deafness and some types of colon
cancer. Using the CELERA Database, we were able to decipher the genomic
organization of the ERdj5 gene. It spans ~62 kb and is
composed of 24 exons (Fig. 2B). Splicing variants were
detected both in ESTs and in cDNAs. Homologs of human
ERdj5 are present in both Mus musculus and
Caenorhabditis elegans (Fig. 1).
Tissue Distribution of ERdj5--
Human multiple-tissue Northern
blots were used to determine transcript size and distribution using the
ERdj5 ORF as a probe. Signals were detected in all tissues
analyzed; their distribution was similar to that of PDI mRNAs,
being particularly abundant in secretory organs (Fig.
3). Multiple ERdj5 bands,
possibly representing splicing variants, were identified in the
pancreas and testis, with bands of less intensity identified in the
heart, liver, spleen, prostate, and ovary and weaker bands seen in the
rest of the tissue panel. A similar pattern was confirmed using the
virtual tissue distribution
program.2
Expression of ERdj5 in Secretory Tissues--
In situ
hybridization was employed to further analyze the distribution of
ERdj5 transcripts in mice. The results shown in Fig.
4 confirm that ERdj5
transcripts are particularly abundant in secretory tissues. Thus, the
adrenal gland showed very strong signals in the cortex and medulla
(Fig. 4A). Expression in the nervous system was variable.
Strong signals were found in several areas of the central nervous
system, particularly in the hippocampus and the granular cell layer of
the cerebellar cortex (Fig. 4B). Moderate signals were seen
in the cerebral cortex, olfactory bulb, striatum, hypothalamus, and
brain stem, whereas the thalamus showed lower expression. White matter
was negative. A strong signal was present in the ependyma covering the
ventricles and plexus choroideus (Fig. 4B). In the spinal
cord, there was a moderate signal in the gray matter, but the signal
was absent in the white matter. In the peripheral nervous system, a
strong signal was evident in the sympathetic (superior cervical) and
sensory (trigeminal) ganglia (Fig. 4C). From emulsion
autoradiography, it was apparent that the signal was present in the
neurons, but absent in the glial cells (Fig. 4D).
ERdj5 mRNAs were abundant in genital organs. The
epididymis showed a strong signal in the epithelium (Fig. 4E). The prostate expressed ERdj5 at the highest
level of all organs studied (Fig. 4F). In dipped
sections, the signal was restricted to the epithelium (Fig.
4G). Also the alimentary tract expressed ERdj5 in
several areas. In the tongue, the stratified epithelium showed moderate
labeling, whereas the striated muscle was not labeled (Fig.
4H). In the oral cavity, the epithelium and sublingual salivary glands were strongly labeled (Fig. 4I). In the
esophagus (Fig. 4J) and larynx (Fig. 4K), the
signal was particularly intense in the mucosa and mucous glands,
respectively. Throughout the intestinal canal, from the stomach to the
colon, a strong signal was seen in the mucosa, whereas the smooth
muscle layer was negative (Fig. 4L).
ERdj5 Is Localized to the ER--
The presence of an N-terminal
hydrophobic sequence and a C-terminal KDEL motif suggests that ERdj5 is
an ER-resident protein. Indeed, a chimeric protein in which GFP was
inserted immediately before the KDEL sequence of ERdj5 displayed a
reticular pattern, largely coinciding with the ER marker used,
pDsRed-ER (Fig. 5). Also, Myc-tagged
ERdj5 was localized in the ER of HeLa
transfectants.3
ERdj5 Interacts with BiP via the DnaJ Domain--
Hsp70 members
have been shown to interact in vitro with GST fusion
proteins containing their J domain protein partners (2, 25, 30-32). To
investigate whether the DnaJ domain of ERdj5 can interact with BiP, we
used surface plasmon resonance assays (33). Either full-length ERdj5 or
its DnaJ domain fused with GST was immobilized in separate channels on
a BIAcore CM5 chip (see "Experimental Procedures"). Binding to BiP
was then tested in the presence of ATP and ADP. In the presence of ATP,
the BiP protein bound to both GST-DnaJ and GST-ERdj5. No binding to the
control channel containing the GST protein alone was observed. As
expected, there was no binding in the presence of ADP or in the absence
of either ATP or ADP. To further confirm the binding of ERdj5 and its
DnaJ domain to BiP, in vitro GST pull-down experiments were
carried out (Fig. 6, B and
C). In vitro translated BiP bound to GST-DnaJ and
GST-ERdj5, but not to GST (Fig. 6B). Likewise, both
GST-ERdj5 and GST-DnaJ bound to purified His-tagged BiP. Binding was
more efficient in the presence of ATP (Fig. 6C).
ERdj5 Is Up-regulated upon ER Stress--
The accumulation of
unfolded proteins in the ER activates the UPR, thus inducing the
transcription of many ER-resident chaperones. We examined whether ERdj5
is also up-regulated in times of ER stress. Following treatment of
HEK293T cells with a panel of common UPR inducers as previously
described (34, 35), ERdj5 transcription was up-regulated (Fig.
7). Heat shock and serum deprivation
resulted in no induction, suggesting that ERdj5 is a
bona fide UPR gene. This correlates with the presence of an
ERSE-like box (CCAATN9CGCGG) ~330 bases upstream of the
ERdj5 ATG start codon, with N9 consisting of
mainly guanine and cytosine nucleotides, which is common for ERSE
motifs. The ERdj5 pattern of up-regulation following stress is similar
to that seen for Ero1-L We describe here a novel human ER-resident protein (ERdj5)
ubiquitously expressed and abundant in secretory tissues such as pancreas and testis. Homologs of human ERdj5 are present in
both M. musculus and C. elegans. The presence of
DnaJ-, PDI-, and thioredoxin-like domains suggests that ERdj5 may be
involved in assisting protein folding and quality control in the ER.
Several lines of evidence including the transcriptional regulation of
ERdj5 and its subcellular localization and molecular
interactions support this hypothesis. The presence of an N-terminal
leader sequence and a C-terminal KDEL motif confirm our findings that
ERdj5 is localized in the ER. GFP-tagged ERdj5 with the ER retention
signal KDEL yields a reticular staining pattern, largely superimposable
on that obtained with ER markers.
As in the case of many other proteins induced during the UPR, the
ERdj5 promoter contains a putative ERSE box (9). This correlates with the observation that tunicamycin, dithiothreitol, and
thapsigargin and other common UPR inducers, but not cell stress inducers such as heat shock, UV light, and serum deprivation, induced
the accumulation of ERdj5 transcripts in HEK293T cells. The
fact that ERdj5 transcripts were not so highly induced could reflect the presence of a single ERSE-like box, whereas other ER
chaperones such as BiP have multiple ERSE boxes and are usually very
highly induced upon ER stress (9, 19, 34). In HEK293T cells, all
ERdj5 transcripts were induced to a lesser extent than Ero1-L DnaJ proteins normally work in tandem with an Hsp70 partner,
stimulating cycles of ATP hydrolysis and thus allowing Hsp70 proteins to bind and release substrates (36, 37). The human ER-resident Hsp70 protein BiP is no exception. Two DnaJ domain proteins
(HEDJ and ERdj4) have recently been shown to bind to BiP and to
stimulate its ATPase activity in vitro, respectively (25,
28). Our results show that also ERdj5 could be involved in this folding
cycle via an interaction with BiP. Indeed, either full-length ERdj5 or
its DnaJ domain alone can bind BiP in an ATP-dependent
manner. Lack of ATP or substitution with ADP resulted in little or no
interaction, suggesting that ERdj5 could function as a DnaJ cofactor
for BiP.
The most intriguing feature of ERdj5 is the presence of DnaJ-,
thioredoxin-like, and PDI-like domains on the same polypeptide backbone. Why does ERdj5 contain domains posed for different redox functions? And why are they linked to a DnaJ domain? The majority of
membrane and secretory cargo proteins contain disulfide bonds that are
often essential to attain the native state. Disulfide bond formation
and disulfide isomerization occur in the ER and are catalyzed by an
array of resident oxidoreductases, the prototype of which is PDI. In
addition, disulfide bonds are reduced in the ER lumen prior to the
dislocation of proteins destined to degradation (38, 39). Redox
regulation in the ER must hence be precisely orchestrated (21). PDI
functions as a catalyst in the oxidation and isomerization of
disulfides on nascent polypeptides undergoing folding in the oxidizing
environment of the ER (1). Recently, this oxidoreductase has also been
involved in the dislocation of short-lived proteins targeted to
proteasomal destruction (40, 41). Members of the thioredoxin/PDI
families invariably contain a CXXC motif. The redox activity
of these proteins is dictated by the local pK of the relevant thiols
groups, which in turn depends on the flanking amino acids. Thioredoxin,
which normally acts as a reductase in the cytosol, can be turned into
an oxidase by transplanting the intervening amino acids with those
normally found in PDI (42, 43).
An important question remains concerning ERdj5: will it work as a
thioredoxin, a PDI-like protein, or simply as a DnaJ partner for a
corresponding Hsp70 or as a combination of all three? Using the
standard thioredoxin activity assays of insulin reduction (44, 45), we
have so far been unable to show any activity for recombinant ERdj5
(data not shown). However, this does not exclude that ERdj5 can act as
a reductase on other substrates. Further analysis is ongoing to
determine the thioredoxin activity of each of the four CXXC domains.
There are at least two main folding pathways that a nascent
glycoprotein may select upon entry into the ER. One utilizes calnexin and calreticulin as chaperones; the other is based primarily on BiP.
Calnexin and calreticulin have been shown to associate with ERp57, an
oxidoreductase that shares extensive similarities with PDI (46), but
does not seem to utilize Ero1 to be reoxidized (47). ERp57 plays a key
role in the folding of class I major histocompatibility complex
molecules (48). The calnexin-ERp57 partnership provides a dual
specialization to nascent proteins that engage in this pathway, the
former acting as a chaperone and the latter as an oxidoreductase. How
does the BiP pathway handle the making of disulfide bonds in its client
proteins? In yeast, BiP and PDI seem to interact with each other (49),
perhaps forming a complex not dissimilar from the calnexin-ERp57 duo. The interaction we detected between BiP and the ERdj5 DnaJ domain is
bound to bring the redox active domains of the latter in the vicinity
of the main ER chaperone. Furthermore, a cooperative relationship
between BiP and PDI has previously been shown in vitro in
the oxidative folding of antibodies (50). It was hypothesized that in
the absence of BiP, unfolded antibody chains collapse rapidly upon
refolding, leaving the cysteine side chains inaccessible to PDI; but
that in the presence of BiP, BiP binds the unfolded polypeptide chains
and keeps them in a suitable conformation for PDI accessibility (50).
Perhaps this is also the case for ERdj5. Its DnaJ domain could
stimulate the ATPase activity of BiP, allowing BiP to bind unfolded
polypeptide, thus leaving the cysteine residues of the polypeptide
available for ERdj5.
Proteins that do not mature properly are retained in the ER and are
eventually targeted for ER-associated degradation through the action of
chaperones (51). Release from BiP is essential for substrates to
attempt productive folding as well as to be dislocated across the ER
membrane for proteasomal destruction. During acute ER stress,
detachment of substrates from chaperones may cause protein aggregation.
In yeast, however, the UPR is intimately connected with the pathways of
ER-associated degradation (7, 52). The thioredoxin-like domain present
in ERdj5 might provide the reductive force that seems to be necessary
to complete unfolding of proteins targeted for dislocation. It may also
be important in reducing PDI so as to allow its function as an
isomerase and in promoting dislocation (40). On the other hand, the
additional domains (more similar to PDI) may enable ERdj5 to act on a
wider range of substrates.
Despite the control mechanisms that exist in the ER, there are many
diseases that are attributed to the misfolding or aggregation of
proteins within the ER, e.g. Alzheimer's disease,
prion-associated disorders, cystic fibrosis, etc. (53-56). ERdj5 is
expressed in the neuronal cells of the hippocampus (in situ
data not shown), which is a site of neuron degeneration in the brains
of Alzheimer's disease patients. Perhaps ERdj5 also has a role in
these misfolding disease states. Determining the precise function of
ERdj5 and establishing whether the BiP-ERdj5 duo is important in the
folding pathway or in the unfolding that is a prelude to dislocation
deserve further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside and incubating for
an additional 4 h at 37 °C. Cells were pelleted by
centrifugation. The pellet was resuspended in 12.5 ml of ice-cold buffer containing 20 mM Tris (pH 8), 50 mM
NaCl, and 1 mM phenylmethylsulfonyl fluoride. 150 µl of
lysozyme (50 mg/ml) was added, and the solution was left stirring on
ice for 30 min. Bacteria were then placed on ice, and 0.1 M
MnCl2, 1 M MgCl2, 1 mg/ml DNase,
and 1 mg/ml RNase (150 µl each) were added and incubated for a
further 45 min on ice. Bacteria were lysed by sonication, and the
debris was removed by centrifugation at 15,000 rpm for 45 min at
4 °C. The supernatant was added to 2 ml of prepared glutathione
slurry, and protein was eluted according to the manufacturer's
recommendations (Amersham Biosciences).
-32P]dCTP (Rediprime random primer labeling kit,
Amersham Biosciences) and hybridized at 65 °C overnight in
ExpressHyb solutions following the protocol provided by
Clontech. The blots were also hybridized with human
-actin as a control.
20 °C until used. Four oligonucleotide
probes for the ORF of mouse ERdj5 (nucleotides 329-356,
543-582, 1269-1303, and 2232-2269, GenBankTM/EBI
accession number AF255459) were used and labeled to a specific activity
of 1 × 109 cpm/µg with
[
-33P]dATP (PerkinElmer Life Sciences) using
terminal deoxynucleotidyltransferase (Amersham Biosciences). The probe
sequences exhibit <60% similarity to all known sequences in the
GenBankTM/EBI Data Bank. All probes gave similar expression
results when used separately and were usually combined to intensify the
hybridization signal. As a control for the specificity of in
situ hybridizations, several probes for unrelated mRNAs
with known expression patterns and with similar lengths and GC contents
were used. The addition of a 100-fold excess of the respective
unlabeled probe abolished all hybridization signals. In situ
hybridization was carried out as described previously (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (87K):
[in a new window]
Fig. 1.
Homology alignment of human ERdj5 with
C. elegans and M. musculus
ERdj5. Shown is a comparison of the derived human ERdj5
sequence with the M. musculus and C. elegans
homologs identified in the M. musculus and C. elegans genome data bases, respectively. Gray boxes
indicate amino acid matches. Within the protein sequence of ERdj5, the
tripeptide His-Pro-Asp conserved in all DnaJ proteins is surrounded
with a single-lined box, and the four putative thioredoxin
domains are highlighted with double-lined boxes.
View larger version (29K):
[in a new window]
Fig. 2.
Genomic and domain organization of ERdj5.
A, ERdj5 is an ER-resident protein containing DnaJ-,
thioredoxin (Trx)-, and PDI-like domains. In the scheme,
DnaJ proteins are classified based on the presence of a J domain, a
G/F-rich region, and a cysteine-rich region. Classification of
thioredoxin-like domains is based on sequence similarity of their
putative active sites to the thioredoxin or PDI CXXC motif.
B, genomic organization of human the ERdj5 gene.
The 24 exons of human ERdj5 (represented by numbered
gray and black bars) are distributed over ~62 kb of
chromosome 2 at p22.1-23.1. Exons 1-3 (gray bars) are
composed of the 5'-untranslated region, with the ORF starting in exon
3. Exon 24 (gray bar) contains the stop codon plus the
3'-untranslated region.
View larger version (54K):
[in a new window]
Fig. 3.
Detection of ERdj5 mRNA
expression in human tissues by RNA hybridization. A blot
containing 2 µg of polyadenylated RNA from human tissues was
hybridized with radiolabeled ERdj5 cDNA, PDI cDNA,
or -actin probes. The migration positions of molecular mass markers
are indicated. P.B.L., peripheral blood leukocytes.
View larger version (153K):
[in a new window]
Fig. 4.
Demonstration of ERdj5
mRNA in mouse tissues by in situ
hybridization. A, strong signals for
ERdj5 are seen in the cortex (co) and medulla
(me) of the adrenal gland. B, the sagittal
section of the brain shows strong signals in the hippocampus
(hc), the granular layer of the cerebellar cortex
(gcl), and the ependyma (ep) in ventricles.
Moderate expression is seen in the olfactory bulb (ob),
cerebral cortex (co), striatum (str), hypothalamus
(ht), and brain stem (bs). A weak
signal is present in the thalamus (th), whereas the white
matter (corpus callosum indicated by arrowheads) is not
labeled. C, a strong signal is shown in the neurons of
trigeminal ganglia (arrows), but is absent in glial cells.
D, a dipped section shows a signal in the neurons of
the trigeminal ganglion (arrowheads). Bar = 40 µm. E, in the epididymis, a strong signal is present in
the epithelium (arrows), whereas stroma
(arrowheads) is unlabeled. F, an
extremely strong signal is seen in the epithelium of the prostate
(arrows), whereas the stroma (arrowheads) is
negative. G, a dipped section shows a strong signal
in the epithelium (ep) of the prostate, whereas stromal
(st) fibroblasts and smooth muscle cells are unlabeled.
Bar = 15 µm. H, the stratified squamous
epithelium (arrows) of the tongue is clearly labeled,
whereas striated muscle (mu) is unlabeled. I, in
the floor of the oral cavity, the epithelium (arrows) and
the sublingual salivary glands (arrowheads) are intensely
labeled. mu, muscle. J, the epithelium of the
esophagus (arrows) is labeled, whereas the smooth muscle
layer (mu) is negative. K, the mucous glands of
the larynx (arrows) are strongly positive, whereas the
thyroid glands (arrowheads) are weakly labeled, and the
muscles (mu) are unlabeled. L, a strong signal is
seen in the mucosa (arrows) of the colon, whereas the muscle
layer (arrowheads) is unlabeled.
View larger version (47K):
[in a new window]
Fig. 5.
ERdj5 is an ER-resident protein.
A, HEK293 cells were transiently transfected with
pEGFP-ERdj5, encoding the ORF of ERdj5 fused to GFP with the
endogenous ER KDEL at the C terminus. B, pDsRed2-ER
(Clontech) was used as an ER localization control.
C, the merging of A and B shown.
View larger version (30K):
[in a new window]
Fig. 6.
ATP-dependent binding of ERdj5 to
BiP. A, BIAcore interaction studies of BiP with ERdj5 and
its DnaJ domain. BiP was diluted to a final concentration of 100 µM in HBS-EP supplemented with 5 mM
MgCl2 and either 5 mM ATP (solid
lines) or 5 mM ADP (broken lines) and
immediately analyzed for binding to DnaJ (red) and ERdj5
(blue) by surface plasmon resonance. B, in
vitro translated BiP binds to ERdj5 and the DnaJ domain. GST
pull-down experiments were performed to show the interaction between
purified GST-ERdj5 or GST-DnaJ and in vitro translated BiP
protein. Asterisks indicate GST-tagged proteins. The
first lane contains 10% of the input of in vitro
translated BiP that was added to the GST-tagged proteins. The
second through fourth lanes contain GST
protein and GST-tagged protein without the addition of in
vitro translated BiP. The fifth through seventh
lanes contain in vitro translated BiP added to the GST
and GST-tagged proteins. C, ATP facilitates the binding of
purified His-tagged BiP to the DnaJ domain of ERdj5. Purified
His-tagged BiP (2 µg) was incubated with GST, GST-DnaJ, or GST-ERdj5
in the presence of 2 mM ATP or ADP as indicated. The
seventh lane shows 4% of the His-tagged BiP input.
, which is a known UPR
gene (35).
View larger version (78K):
[in a new window]
Fig. 7.
ERdj5 transcripts are up-regulated
upon ER stress. HEK293T cells were treated with a number of
stress-inducing drugs and conditions. Blots were hybridized with probes
specific for ERdj5, Ero1-L (35), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as
indicated. Arrows indicate possible splicing variants.
DTT, dithiothreitol; DOG, deoxyglucose;
FCS, fetal calf serum.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or BiP (34-35). The
physiological significance of this difference deserves further investigation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Cristina Benedetti and Ineke Braakman for helpful reagents and suggestions. The excellent technical assistance of Ulla-Margit Jukarainen is greatly appreciated.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Swedish Medical Research Council Projects 13X-10370 and 19X-11622-03C and by grants from the Medical Research Fund of Tampere University Hospital, the Funds of University of Tampere, the Karolinska Institute, Södertörns Högskola, the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Italian Ministry of University and Research (MIUR, Center of Excellence in Physiopathology of Cell Differentiation), PRIN (2002.058218_006), and Telethon (GP0117/01). The CELERA Database was used under, license agreement with the Karolinska Institute (Stockholm).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF038503 and AF255459.
§ Both authors contributed equally to this work.
Recipient of a fellowship from Telethon (380/bs).
¶¶ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
46-8-608-9162; Fax: 46-8-774-5538; E-mail:
giannis.spyrou@cbt.ki.se.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M206995200
2 Available at www.labonweb.com.
3 T. Simmen, G. Bertoli, and R. Sitia, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; PDI, protein-disulfide isomerase; UPR, unfolded protein response; ERSE, endoplasmic reticulum stress element; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; ORF, open reading frame; GFP, green fluorescent protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Noiva, R. (1999) Semin. Cell Dev. Biol. 10, 481-493[CrossRef][Medline] [Order article via Infotrieve] |
2. | Brooks, D. A. (1999) Semin. Cell Dev. Biol. 10, 441-442[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Fink, A. L.
(1999)
Physiol. Rev.
79,
425-449 |
4. |
Xiong, X.,
Chong, E.,
and Skach, W. R.
(1999)
J. Biol. Chem.
274,
2616-2624 |
5. | Kopito, R. R. (1997) Cell 88, 427-430[Medline] [Order article via Infotrieve] |
6. | Mori, K. (2000) Cell 101, 451-454[Medline] [Order article via Infotrieve] |
7. | Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J., Weissman, J. S., and Walter, P. (2000) Cell 101, 249-258[Medline] [Order article via Infotrieve] |
8. | Lee, A. S. (2001) Trends Biochem. Sci. 26, 504-510[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Yoshida, H.,
Haze, K.,
Yanagi, H.,
Yura, T.,
and Mori, K.
(1998)
J. Biol. Chem.
273,
33741-33749 |
10. | Kohno, K., Normington, K., Sambrook, J., Gething, M. J., and Mori, K. (1993) Mol. Cell. Biol. 13, 877-890[Abstract] |
11. | Kelley, W. L. (1998) Trends Biochem. Sci. 23, 222-227[CrossRef][Medline] [Order article via Infotrieve] |
12. | Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2874-2878[Abstract] |
13. | Misselwitz, B., Staeck, O., and Rapoport, T. A. (1998) Mol. Cell 2, 593-603[Medline] [Order article via Infotrieve] |
14. | Cheetham, M. E., and Caplan, A. J. (1998) Cell Stress Chaperones 3, 28-36[CrossRef][Medline] [Order article via Infotrieve] |
15. | Ferrari, D. M., and Soling, H. D. (1999) Biochem. J. 339, 1-10[CrossRef][Medline] [Order article via Infotrieve] |
16. | Freedman, R. B., Hirst, T. R., and Tuite, M. F. (1994) Trends Biochem. Sci. 19, 331-336[CrossRef][Medline] [Order article via Infotrieve] |
17. | Frand, A. R., and Kaiser, C. A. (1999) Mol. Cell 4, 469-477[Medline] [Order article via Infotrieve] |
18. |
Tu, B. P., Ho-,
Schleyer, S. C.,
Travers, K. J.,
and Weissman, J. S.
(2000)
Science
290,
1571-1574 |
19. |
Anelli, T.,
Alessio, M.,
Mezghrani, A.,
Simmen, T.,
Talamo, F.,
Bachi, A.,
and Sitia, R.
(2002)
EMBO J.
21,
835-844 |
20. | Sun, X., Dai, Y., Liu, H., Chen, S., and Wang, C. (2000) Biochim. Biophys. Acta 1481, 45-54[Medline] [Order article via Infotrieve] |
21. | Fassio, A., and Sitia, R. (2002) Histochem. Cell Biol. 117, 151-157[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
23. | Frangioni, J. V., and Neel, B. G. (1993) Anal. Biochem. 210, 179-187[CrossRef][Medline] [Order article via Infotrieve] |
24. | Kozak, M. (1996) Mamm. Genome 7, 563-574[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Yu, M.,
Haslam, R. H.,
and Haslam, D. B.
(2000)
J. Biol. Chem.
275,
24984-24992 |
26. | Konenen, J., and Pelto-Huikko, M. (1997) Technical Tips Online http://www.tto.trends.com/ |
27. | Cabibbo, A., Consalez, G. G., Sardella, M., Sitia, R., and Rubartelli, A. (1998) Oncogene 16, 2935-2943[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Shen, Y.,
Meunier, L.,
and Hendershot, L. M.
(2002)
J. Biol. Chem.
277,
15947-15956 |
29. | Cyr, D. M., Langer, T., and Douglas, M. G. (1994) Trends Biochem. Sci. 19, 176-181[CrossRef][Medline] [Order article via Infotrieve] |
30. | Holstein, S. E., Ungewickell, H., and Ungewickell, E. (1996) J. Cell Biol. 135, 925-937[Abstract] |
31. |
Misselwitz, B.,
Staeck, O.,
Matlack, K. E.,
and Rapoport, T. A.
(1999)
J. Biol. Chem.
274,
20110-20115 |
32. |
Suh, W. C., Lu, C. Z.,
and Gross, C. A.
(1999)
J. Biol. Chem.
274,
30534-30539 |
33. | Fagerstam, L. G., Frostell, A., Karlsson, R., Kullman, M., Larsson, A., Malmqvist, M., and Butt, H. (1990) J. Mol. Recognit. 3, 208-214[Medline] [Order article via Infotrieve] |
34. | Benedetti, C., Fabbri, M., Sitia, R., and Cabibbo, A. (2000) Biochem. Biophys. Res. Commun. 278, 530-536[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Pagani, M.,
Fabbri, M.,
Benedetti, C.,
Fassio, A.,
Pilati, S.,
Bulleid, N. J.,
Cabibbo, A.,
and Sitia, R.
(2000)
J. Biol. Chem.
275,
23685-23692 |
36. |
Nishikawa, S.,
and Endo, T.
(1997)
J. Biol. Chem.
272,
12889-12892 |
37. |
Corsi, A. K.,
and Schekman, R.
(1997)
J. Cell Biol.
137,
1483-1493 |
38. |
Tortorella, D.,
Story, C. M.,
Huppa, J. B.,
Wiertz, E. J.,
Jones, T. R.,
Bacik, I.,
Bennink, J. R.,
Yewdell, J. W.,
and Ploegh, H. L.
(1998)
J. Cell Biol.
142,
365-376 |
39. |
Fagioli, C.,
Mezghrani, A.,
and Sitia, R.
(2001)
J. Biol. Chem.
276,
40962-40967 |
40. | Tsai, B., Rodighiero, C., Lencer, W. I., and Rapoport, T. A. (2001) Cell 104, 937-948[Medline] [Order article via Infotrieve] |
41. | Tsai, B., Ye, Y., and Rapoport, T. A. (2002) Nat. Rev. Mol. Cell. Biol. 3, 246-255[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Krause, G.,
Lundstrom, J.,
Barea, J. L.,
Pueyo de la Cuesta, C.,
and Holmgren, A.
(1991)
J. Biol. Chem.
266,
9494-9500 |
43. |
Mossner, E.,
Huber-Wunderlich, M.,
and Glockshuber, R.
(1998)
Protein Sci.
7,
1233-1244 |
44. |
Spyrou, G.,
Enmark, E.,
Miranda-Vizuete, A.,
and Gustafsson, J.-Å
(1997)
J. Biol. Chem.
272,
2936-2941 |
45. |
Wollman, E. E.,
d'Auriol, L.,
Rimsky, L.,
Shaw, A.,
Jacquot, J. P.,
Wingfield, P.,
Graber, P.,
Dessarps, F.,
Robin, P.,
and Galibert, F.
(1988)
J. Biol. Chem.
263,
15506-15512 |
46. |
Freedman, R. B.,
Klappa, P.,
and Ruddock, L. W.
(2002)
EMBO Rep.
3,
136-140 |
47. |
Mezghrani, A.,
Fassio, A.,
Benham, A.,
Simmen, T.,
Braakman, I.,
and Sitia, R.
(2001)
EMBO J.
20,
6288-6296 |
48. | Dick, T. P., Bangia, N., Peaper, D. R., and Cresswell, P. (2002) Immunity 16, 87-98[Medline] [Order article via Infotrieve] |
49. |
Gillece, P.,
Luz, J. M.,
Lennarz, W. J.,
de la Cruz, F. J.,
and Romisch, K.
(1999)
J. Cell Biol.
147,
1443-1456 |
50. |
Mayer, M.,
Kies, U.,
Kammermeier, R.,
and Buchner, J.
(2000)
J. Biol. Chem.
275,
29421-29425 |
51. |
Ellgaard, L.,
Molinari, M.,
and Helenius, A.
(1999)
Science
286,
1882-1888 |
52. | Friedlander, R., Jarosch, E., Urban, J., Volkwein, C., and Sommer, T. (2000) Nat. Cell. Biol. 2, 379-384[CrossRef][Medline] [Order article via Infotrieve] |
53. | Taguchi, J., Fujii, A., Fujino, Y., Tsujioka, Y., Takahashi, M., Tsuboi, Y., Wada, I., and Yamada, T. (2000) Acta Neuropathol. 100, 153-160[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Chai, Y.,
Koppenhafer, S. L.,
Bonini, N. M.,
and Paulson, H. L.
(1999)
J. Neurosci.
19,
10338-10347 |
55. | Katayama, T., Imaizumi, K., Sato, N., Miyoshi, K., Kudo, T., Hitomi, J., Morihara, T., Yoneda, T., Gomi, F., Mori, Y., Nakano, Y., Takeda, J., Tsuda, T., Itoyama, Y., Murayama, O., Takashima, A., St., George-Hyslop, P., Takeda, M., and Tohyama, M. (1999) Nat. Cell. Biol. 1, 479-485[CrossRef][Medline] [Order article via Infotrieve] |
56. | Gething, M. J. (2000) Nat. Cell. Biol. 2, E21-E23[Medline] [Order article via Infotrieve] |