From the Research and Education Center for Genetic Information, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Received for publication, August 15, 2002, and in revised form, November 8, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several endoplasmic reticulum (ER)-resident
luminal proteins have a characteristic ER retrieval signal, KDEL, or
its variants at their C terminus. Our previous work searching EST
databases for proteins containing the C-terminal KDEL motif predicted
some novel murine proteins, one of which designated JPDI
(J-domain-containing protein
disulfide isomerase-like protein) is
characterized in this study. The primary structure of JPDI is unique,
because in addition to a J-domain motif adjacent to the N-terminal
translocation signal sequence, four thioredoxin-like motifs were found
in a single polypeptide. As examined by Northern blotting, the
expression of JPDI was essentially ubiquitous in tissues and almost
independent of ER stress. A computational prediction that JPDI is an
ER-resident luminal protein was experimentally supported by
immunofluorescent staining of epitope-tagged JPDI-expressing cells
together with glycosylation and protease protection studies of this
protein. JPDI probably acts as a DnaJ-like partner of BiP, because a
recombinant protein carrying the J-domain of JPDI associated with BiP
in an ATP-dependent manner and enhanced its ATPase
activity. We speculate that for the folding of some proteins in the ER,
chaperoning by BiP and formation of proper disulfide bonds may
synchronously occur in a JPDI-dependent manner.
Most eukaryotic membrane or secretory proteins are thought to be
folded in the endoplasmic reticulum
(ER).1 Glycosylation and
disulfide bond formation are involved in the correct folding of these
proteins because inhibition of such post-translational modifications
causes accumulation of unfolded proteins in the ER. As described
below, various molecular chaperones and folding enzymes located
in the ER play crucial roles in these cellular events (1).
BiP, also known as GRP78, is an ER-resident member of the Hsp70
chaperone family. As reviewed by Gething (2), it is apparent that in
the ER, many unfolded proteins are chaperoned by BiP. Through the
maintenance of solubility by BiP, unfolded proteins are occasionally
subjected to ER-associated degradation (3-5). Furthermore,
translocation of newly synthesized proteins into the ER requires BiP,
and it seems that BiP plays multiple roles in the protein translocation
machinery (6-8). In addition, Ire1 and PERK, sensors for
accumulation of unfolded proteins in the ER, are negatively regulated
by BiP (9, 10). Similar to other Hsp70 family chaperones, BiP carries
an N-terminal ATPase domain and an adjacent substrate-binding domain.
The biochemical properties of these domains in Hsp70 family chaperones
are well documented (11). ATP binding to the ATPase domain causes low
affinity but rapid binding of chaperone substrates to the
substrate-binding domain, whereas subsequent ATP hydrolysis stabilizes
substrate binding.
ATPase activity of Hsp70 family chaperones is regulated by various
co-chaperones, including J-proteins (11, 12). The defining feature of
J-proteins is an ~70-amino acid-residue signature termed the
J-domain. Through this domain, J-proteins interact with their Hsp70
chaperone partners to stimulate ATP hydrolysis. Up until now, several
types of ER-resident J-proteins have been identified. Over the
full-length of the molecules, yeast Saccharomyces cerevisiae Scj1 and mammalian HEDJ (13) share common structural features with bacterial DnaJ, which has an intrinsic ability to bind some unfolded proteins and assists in chaperoning by its Hsp70 partner DnaK
(11, 14). It has been demonstrated experimentally that Scj1 and another
J-protein, Jem1, act to chaperone unfolded proteins in the yeast ER (5,
15). In contrast, yeast Sec63 (16), its putative mammalian orthologue
hSec63 (17), and mammalian MTJ1 (18, 19), all of which are
transmembrane J-proteins, seem to be directly involved in protein
translocation across the ER membrane. Their similarity to bacterial
DnaJ is restricted to the J-domain. In addition, mammalian cells
contain a fourth ER-resident J-protein termed ERdj4 (20).
Another distinct family of ER-resident proteins, of which protein
disulfide isomerase (PDI) is best documented (21, 22), is defined by
the presence of thioredoxin-like motifs in their primary structure. PDI
promotes oxidative folding of secretory proteins through catalyzing
thiol-disulfide exchange reactions including disulfide formation,
disulfide reduction, and disulfide isomerization. In this case, the
thioredoxin-like motifs act as the catalytic active centers of PDI
(23). Furthermore, it is widely accepted that PDI acts not only as an
enzyme but also as a chaperone, and in this case, Tsai et
al. (24) proposed that the thioredoxin-like motifs play a
regulatory role.
BiP, PDI, and several other soluble proteins resident in the ER carry a
conserved tetrapeptide sequence, KDEL, at their C terminus (25). This
KDEL motif and its variants are termed the ER retrieval signal, and it
is commonly accepted that proteins carrying this signal are bound by a
receptor in the Golgi apparatus and that the receptor-ligand complex
returns to the ER (26). We previously searched EST databases for
proteins carrying this C-terminal KDEL motif (27). This data base
search predicted some novel proteins, one of which was named EP58 and
is characterized in our previous report (27). Here we describe studies
on another protein, JPDI (J-domain-containing
PDI-like protein), which is a new type of ER-resident
J-protein carrying thioredoxin-like motifs.
Plasmids--
As described previously (27), murine total RNA was
subjected to reverse transcriptase-PCR using an oligo(dT) primer
for the reverse transcription. For the PCR, the primer set
5'-CCGGAATTCCACCATGGGAGTCTGGTTAAAGATGAC-3' and 5'-CCGGAATTCGATCTTCTGATGCCGTTCTCC-3' was employed. Both
primers are specific to the predicted sequence of JPDI with the
exception of additional EcoRI sites (italics)
plus CCG and a Kozak sequence (underlined) (28). The
resulting reverse transcriptase-PCR product carrying the JPDI cDNA
was digested by EcoRI and inserted into the EcoRI
site of pBluescript II KS+ (Stratagene) to obtain plasmid pJPDI-1.
The JPDI cDNA in pJPDI-1 was modified by insertion of a DNA
sequence encoding a hemagglutinin (HA) epitope tag and subcloned into a
mammalian expression vector pCAGGS (29). The resulting plasmid,
pCAG-JPDI-HA, was used for mammalian expression of JPDI tagged with a
HA epitope tag at a position just N-terminally adjacent to the
C-terminal KDEL (JPDI-HA) (see Fig. 1A).
The J-domain-encoding sequence was PCR-amplified from pJPDI-1 using the
primer set 5'-CGCGGATCCGAACAGATCAGAATTTTTACAGTTTACTTGG-3' and
5'-CCGGAATTCTCAATGGTGGTGATGATGGTGGATGAAGAGGCTGGGGTAGC-3'. Both are specific to JPDI with the exception of additional
BamHI and EcoRI sites (italics) plus
CGC or CCG and a (His)6 tag-encoding sequence
(underlined). After digestion with BamHI and
EcoRI, the PCR product was cloned into the corresponding
sites of pBluescript II KS+. The J-domain-encoding fragment was
re-excised from the resulting plasmid by BamH
I/XhoI digestion and inserted into the corresponding sites
of a bacterial expression vector pGEX-5X-2 (Amersham Biosciences) to
obtain plasmid pGEX-J. Plasmid pGEX-J(H63Q) is identical to pGEX-J
except that it carries a mutant version of the J-domain-encoding
sequence -AGTTACAGCCTGATAA- (the mutated nucleotide residue
is underlined).
Cell Culturing and Transfection--
All of the mammalian cells
were cultured in Northern Blot Analysis--
As probes for Northern blot
analysis, the following cDNA fragments were random primer-labeled
with [ Analyses of HA-tagged JPDI Expressed in Mammalian Cultured
Cells--
36 h post-transfection of pCAG-JPDI-HA, HeLa cells were
lysed with 10 mM Tris-HCl (pH 7.5), 10 mM EDTA,
1% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride.
After denaturing of proteins by incubation in 0.5% SDS and 1%
2-mercaptoethanol at 100 °C for 10 min, the lysates were treated
with 50 mM sodium citrate (pH 5.5),
endo-
Isolation of microsomes and protease protection assays were performed
as described previously (27) with the exception that proteinase K was
used at 1 µg/ml. In both EndoH treatment and protease protection
assays, the resulting samples were subjected to Western blot analysis
using the following materials: 12CA5 anti-HA monoclonal antibody
(Nippon Boehringer Ingelheim, Tokyo, Japan) and rabbit anti-human BiP
antibody as primary antibodies; horseradish peroxidase-conjugated
donkey anti-mouse IgGs (Dako) and anti-rabbit IgGs (Amersham
Biosciences) as secondary antibodies; and ECL Western blotting
detection system (Amersham Biosciences). Immunofluorescent staining of
cultured cells was performed as described previously (30).
Preparation of Recombinant Proteins--
N-terminal GST and
C-terminal (His)6 double-tagged JPDI J-domain and its H63Q
mutant version (designated GST-J and GST-J(H63Q), respectively) were
expressed from pGEX-J and pGEX-J(H63Q), respectively. Escherichia
coli BL21-CodonPlus (DE3)-RIL (Stratagene) cells transformed with
these plasmids were cultured in LB broth containing 50 µg/ml ampicillin at 37 °C to A600 1.2. The
cultures were then shifted to 20 °C, and
isopropyl-1-thio-
Unfused GST was obtained from pGEX-5X-2 in E. coli
BL21-CodonPlus (DE3)-RIL by culturing cells in the presence of 1 mM isopropyl-1-thio-
An E. coli strain expressing (His)6-tagged
hamster BiP not containing the translocation signal sequence was a kind
gift of Dr. L. M. Hendershot (St. Jude Children's Research
Hospital, Memphis, TN). The recombinant BiP protein was purified
as described previously (31) and dialyzed against buffer B88 (the same
composition as buffer 88lK with the exception of 150 mM KOAc).
GST Pull-down Assay--
Purified proteins (GST-J, GST-J(H63Q),
and unfused GST) were adjusted to the same concentrations with buffer
88lK. 3 µg of each protein then was diluted to 80 µl with binding
buffer B (20 mM HEPES, 100 mM KCl, 5 mM MgCl2, 0.1% Nonidet P-40, 2% (w/v) glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (pH 6.8)), and 20 µl of
a 50% slurry of glutathione-Sepharose equilibrated with binding buffer
B was added. After incubation by rotation for 1 h at 4 °C, the
beads were collected by centrifugation at 3,000 × g
and washed three times with 1 ml of binding buffer B. The protein-bound
beads were incubated with 2 µg of BiP in a final volume of 100 µl
of binding buffer B containing 0 or 1 mM ATP for a 2-h
rotation at 4 °C. The beads were then collected by centrifugation
and washed five times with 1 ml of binding buffer B. Proteins bound to
the beads were eluted by boiling with 30 µl of SDS-PAGE sample
buffer, subjected to SDS-PAGE, and detected by CBB staining of the gel.
ATPase Assay--
ATPase activity of BiP was monitored as
described previously (32) with minor modifications. 1 µg of BiP was
mixed with GST-J, GST-J(H63Q), or unfused GST in 100 µl of ATPase
assay buffer containing 50 mM HEPES, 50 mM
NaCl, 2 mM MgCl2 (pH 6.8), 100 µM
unlabeled ATP, and 0.5 µCi of [ Sequence Analysis of JPDI--
We previously searched the
GenBankTM data base for DNA sequences corresponding
to KDELter where "ter" means a stop codon, and among the resulting
hits, we have focused on a EST sequence (GenBankTM
accession number AA062302) in this study. Using this EST sequence as
the query, a subsequent BLAST search against the GenBankTM
data base predicted that it is part of a cDNA
(GenBankTM accession number AK004617). This
cDNA, which was generated and identified in the mouse full-length
cDNA encyclopedia project (33), encodes a previously
uncharacterized 793-amino acid-residue protein termed here as JPDI.
Computational analyses using PSORT II (psort.nibb.ac.jp/form2.html) and
Searching Motif (www.motif.genome.ad.jp/) programs indicated as shown
in Fig. 1, A and B,
that JPDI contains several significant sequence features together with
the C-terminal KDEL motif. First, JPDI putatively bears an N-terminal
translocation signal sequence that is cleaved between Gly-31 and
Thr-32. The predicted molecular mass of signal sequence-cleaved JPDI is
calculated as ~87 kDa, which well coincides with
endoglycosidase-treated HA-tagged JPDI from cell lysate (Fig.
3B) as described below. Second, a 66-amino
acid-residue J-domain motif was found adjacent to the translocation
signal sequence. Finally, similar to PDI, JPDI contains multiple
thioredoxin-like motifs (-WCXXC-).
As shown in Fig. 1C, translating the BLAST search against
the GenBankTM data base using the JPDI amino acid sequence
as the query predicted similar uncharacterized proteins in humans (90%
identical) and in Caenorhabditis elegans (37%
identical). These putative orthologues of murine JPDI commonly
carry all of the above mentioned sequence features. No homologue was
found in S. cerevisiae.
Expression of JPDI--
To determine the tissue distribution of
JPDI expression, Northern blot detection of JPDI mRNA was performed
using multi-tissue Northern blot, a commercial Northern blot membrane
on which mouse poly(A)+ RNA preparations from various
tissues were blotted. The JPDI probe almost ubiquitously detected
duplicate bands (4.0 and 3.3 kilobases) in all of the tissues examined
(Fig. 2A). The 4.0-kilobase transcript was major in most of the tissues with the exception that the
3.3-kilobase transcript was highly expressed in the testis. Relatively
speaking, JPDI expression was high in heart, liver, kidney, and testis
and low in spleen and skeletal muscle.
Several mammalian ER-resident chaperones and folding enzymes including
BiP and PDI are transcriptionally induced by ER stress through
intracellular signaling pathways as described previously (34). To
investigate whether the expression of JPDI is induced under ER stress
conditions, NIH3T3 cells were treated with the ER stress inducer
tunicamycin, an N-glycosylation inhibitor, or thapsigargin,
a calcium pump inhibitor. Thereafter, total RNA preparations were
analyzed by Northern blotting, which showed that JPDI mRNA was only
slightly induced (~1.3-fold) under conditions in which BiP mRNA
was induced ~10-fold (Fig. 2, B and C). Similar results were obtained using L tk JPDI Is a ER-resident Luminal Protein--
JPDI carries only one
significant hydrophobic segment, which is positioned at the N terminus
and predicted to be a cleavable translocation signal, on the basis of
its amino acid sequence. Because of this sequence feature and the
C-terminal KDEL motif, JPDI seemed to be an ER-resident luminal
protein. Therefore, we undertook the task of confirming this idea
experimentally by using cultured cells transiently expressing JPDI
tagged with a HA tag at the position just N-terminally adjacent to the
C-terminal KDEL motif (hereafter called JPDI-HA) (see Fig.
1A).
First, JPDI-HA-expressing NIH3T3 cells were doubly immunostained with
anti-HA antibody and anti-PDI antibody. As detected by fluorescence
microscopy, both JPDI-HA staining and PDI staining yielded classical ER
patterns (Fig. 3A,
upper and middle panels). The
yellow/orange color in the overlay image indicates co-localization of
JPDI-HA and PDI (Fig. 3A, lower panel). Similar
images were obtained from JPDI-HA-expressing HeLa and COS7 cells.
Lysates of JPDI-HA-expressing HeLa cells were treated next with EndoH
and subjected to SDS-PAGE followed by Western blot detection of
JPDI-HA. As shown in Fig. 3B, EndoH treatment increased the mobility of JPDI in SDS-PAGE. This result indicates that JPDI is
modified by N-linked glycosylation that is cleavable by EndoH.
Finally, microsomes prepared from JPDI-HA-expressing HeLa cells were
treated with proteinase K, and proteins protected from protease
digestion were detected by Western blot analysis. As shown in Fig.
3C, neither JPDI-HA nor BiP was digested unless the
microsomal membrane was lysed by detergent Trx-100.
The J-domain of JPDI Binds to BiP--
The localization of JPDI to
the ER implies that JPDI may act as a DnaJ-like partner of BiP. This
possibility was tested by experiments designed to observe in
vitro interaction of the JPDI J-domain with BiP. At the beginning
of in vitro analyses, a fusion of GST-JPDI J-domain
(His)6 tag named GST-J was expressed in bacteria and
purified by glutathione affinity and subsequent nickel-chelating chromatography. As monitored by CBB staining after SDS-PAGE separation, contaminating protein was virtually undetectable in the purified preparation of GST-J (Fig.
4A).
A tripeptide motif, HPD, in the J-domain is quite highly conserved
among J-proteins. As initially demonstrated using E. coli DnaJ (35), an amino acid replacement of the HPD motif to QPD causes a
complete loss of J-domain functions. In murine JPDI, the amino acid
residues positioned at 63-65 correspond to this motif (see Fig.
1A). Thus, as a negative control in the following experiments, we generated GST-J(H63Q), a mutant version of GST-J where
the HPD motif is replaced by QPD (see Fig. 1A). An unfused version of GST (hereafter called GST) was also employed as another type
of negative control. Similar to GST-J, CBB staining after SDS-PAGE
separation confirmed that GST-J, GST, and BiP were purified to
homogeneity (Fig. 4A).
For a pull-down experiment to test J-domain binding to BiP,
glutathione-Sepharose beads carrying GST-J, GST-J(H63Q), or GST were
incubated with BiP in the absence or presence of ATP. The subsequent
analysis of pulled-down proteins showed that BiP does not bind with
GST-J(H63Q) or GST but it does with GST-J and that this binding
requires ATP (Fig. 4B).
The J-domain of JPDI Stimulates ATP Hydrolysis by BiP--
It is
well known that the ATPase activity of Hsp70 family chaperones is
enhanced by the J-domain of their specific J-protein partners. Thus, we
next asked whether the J-domain of JPDI stimulates in vitro
ATP hydrolysis by BiP. First, 1 µg of BiP was incubated without
additional protein or with 0.5 µg each of GST, GST-J, or GST-J(H63Q)
in ATPase assay buffer (the molar ratio of BiP to GST-J or GST-J(H63Q)
was 1.4). The results shown in Fig. 5, A and B, indicate that GST and GST-J(H63Q) had
little or no ability to stimulate ATP hydrolysis by BiP and that, on
the contrary, ATPase activity of BiP was enhanced ~2-fold by the
addition of GST-J. This ATP hydrolysis assay was performed next using
various concentrations of GST, GST-J, or GST-J(H63Q), which confirmed that only GST-J stimulates ATP hydrolysis by BiP (Fig.
5C).
Our computational search for proteins bearing a C-terminal ER
retrieval signal predicted a novel murine protein, JPDI. Remarkably, JPDI is the first identified protein carrying both a J-domain and
thioredoxin-like motifs in a single polypeptide chain (Fig. 1). Because
the similarity of JPDI to other J-proteins is restricted to the
J-domain, we suspect that JPDI plays a role(s) quite different from
that of other J-proteins.
According to a further computational data base search, humans and
C. elegans carry putative orthologues of murine JPDI.
The expression of JPDI mRNA was nearly ubiquitous in all the
tissues examined, and ER stressors induced JPDI mRNA expression
only very slightly (Fig. 2). These observations imply that JPDI may
have stress-independent and constitutive function(s) commonly required in metazoan cells. As shown in Fig. 2A, two different sizes
of JPDI mRNA were detected in various murine tissues. We suspect that these transcripts were produced by alternative termination or by
alternative splicing at the 3'-untranslated region, because a 3'-RACE
analysis predicted multiple 3'-untranslated region lengths of JPDI
mRNA (data not shown).
We next experimentally confirmed cellular localization of JPDI to the
ER lumen using mammalian cultured cells transiently expressing an HA
epitope-tagged version of JPDI designated here as JPDI-HA. To avoid
dysfunction of the N-terminal translocation signal sequence and the
C-terminal KDEL motif of JPDI, the HA tag was added not at the N or C
terminus but at the position indicated in Fig. 1A. A
SDS-PAGE mobility shift of JPDI-HA by EndoH digestion indicates the
N-linked glycosylation and thus luminal localization of this
protein (Fig. 3B). We suspect that JPDI is glycosylated at
the N-glycosylation motif NX(S/T) (see Fig.
1A). Moreover, JPDI-HA in a microsomal fraction was
protected from protease digestion (Fig. 3C). This result
strongly suggests luminal localization of the entire region of JPDI.
Localization of JPDI to the ER was demonstrated by anti-HA
immunofluorescent staining, which yielded an ER-like pattern almost
overlapping that of anti-PDI staining (Fig. 3A). Moreover,
the above mentioned susceptibility of JPDI-HA to EndoH digestion also
implies the ER localization of JPDI, because, in general,
N-linked glycosyl chains are modified such that they are not
susceptible to EndoH digestion in the mammalian Golgi apparatus.
Here we also presented two lines of in vitro evidence that
JPDI can interact with BiP as its DnaJ-like partner via the J-domain. First, a GST pull-down assay indicated ATP-dependent
binding of BiP to the JPDI J-domain (Fig. 4). A requirement for
hydrolyzable ATP for the J-protein-Hsp70 chaperone interaction is well
documented (32, 36). The ATPase activity of BiP next was significantly enhanced by the JPDI J-domain (Fig. 5). The following observations indicate that our preparation of JPDI J-domain interacts with BiP as
its functional partner rather than as an unfolded protein substrate,
which also binds to Hsp70 chaperones to stimulate their ATPase activity
(11). First, unlike the case for J-protein partners, the binding of
unfolded protein substrates to Hsp70 chaperones is inhibited by ATP
(36). Second, amino acid replacement of the highly conserved HPD motif
with QPD in the J-domain, which commonly causes dysfunction of various
J-proteins (35, 37), significantly decreased the ability of the JPDI
J-domain to bind to BiP and to stimulate its ATPase activity (Figs. 4
and 5). Considering the high degree of specificity of a J-domain for
its Hsp70 chaperone partner (38), it is likely that BiP is the natural
Hsp70 chaperone partner of JPDI. However, we cannot exclude the
possibility that other ER-resident Hsp70 family proteins including
GRP170 (39) interact with JPDI.
Thus far, we have failed to demonstrate any PDI-like activities for
JPDI, although the multiple thioredoxin-like motifs of JPDI imply its
activity in disulfide formation. Heterologous expression of murine JPDI
was not able to rescue the lethal phenotype of a PDI gene disruption in
S. cerevisiae (data not shown). In our in vitro
assay, oxidative refolding of denatured ribonuclease A was not promoted
by JPDI, and we have not shown so far thiol-dependent reductase activity of recombinant JPDI, which catalyzes the reduction of insulin disulfides by dithiothreitol (data not shown). Nevertheless, if we suppose a disulfide isomerization activity of JPDI, it is possible to speculate that this reaction occurs synergistically with
chaperoning by BiP. In this scenario, BiP may be recruited by the JPDI
J-domain and bind unfolded protein substrates to maintain their form,
thus making them accessible to the JPDI thioredoxin-like catalytic
centers for disulfide isomerization. Cooperation between BiP and PDI
was demonstrated in an in vitro refolding reaction of a
denatured and reduced model protein (40). Moreover, another thioredoxin-like motif-containing ER-resident protein, ERp57, was
proposed to function together with the lectin-like chaperones calnexin
and calreticulin to promote glycoprotein folding (41, 42). The above
mentioned hypothesized function of JPDI would be a novel example of
synergy between a chaperone and a folding enzyme. An alternative
hypothesis for JPDI function, which is also a speculation derived from
the knowledge of PDI, is that JPDI acts to bind with unfolded proteins.
If this idea is correct, it is possible that like bacterial DnaJ JPDI
functions to bring unfolded proteins to its Hsp70 chaperone partner. In
this case, the thioredoxin-like motifs may play a regulatory role in
retrotranslocation from the ER to cytosol as hypothesized (24).
Otherwise, JPDI may somehow help the function of BiP in the protein
translocation machinery. We anticipate that our further studies
including the generation of JPDI-deficient cells will clarify the
biochemical and physiological function(s) of JPDI.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum Eagle's medium (ICN) supplemented with
10% fetal bovine serum at 37 °C and 5% CO2. Transient
transfection of plasmids was performed as described previously
(30).
-32P]dCTP: murine JPDI and murine BiP (entire
coding region); human glyceraldehyde-3-phosphate dehydrogenase (+75 to
+1019); and human
-actin. Mouse multi-tissue Northern blot was
purchased from Clontech. Extraction of total RNA
from cultured cells and Northern blotting were performed as described
previously (30). After hybridization, blots were washed in 2× SSC at
65 °C and exposed to BAS imaging plate (Fuji film, Tokyo, Japan).
-N-acetylglucosaminidase H (EndoH) (a MBP-tagged
version commercially called EndoHf, New England BioLabs),
and 5 units/1 µg of lysate protein at 37 °C.
-D-galactopyranoside was added (final
concentration, 0.1 mM). After further culture for 80 min, cells were harvested and resuspended in lysis buffer
(phosphate-buffered saline containing 2 mM EDTA, 2 mM 2-mercaptoethanol, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 10 µg/ml pepstatin A, 1 mM
phenylmethylsulfonyl fluoride). The cells were then lysed by the
addition of Triton X-100 (Trx-100, final concentration of 1%) and
sonication. The lysates were clarified by centrifugation twice
(9,500 × g for 20 min and 100,000 × g
for 30 min) and then loaded into a 1-ml bed of
glutathione-Sepharose 4B (Amersham Biosciences) column pre-equilibrated
with lysis buffer. The column was washed with 5 ml of lysis buffer, 10 ml of wash buffer A (lysis buffer plus 1 M KCl and 0.1%
Trx-100), and 5 ml of wash buffer B (50 mM Tris-HCl (pH
7.5), 10 mM ATP, 10 mM Mg(OAc)2,
200 mM KOAc used to remove contaminating DnaK). Bound
proteins were eluted with 8 ml of elution buffer A (50 mM
Tris-HCl, 20 mM reduced glutathione (pH 8.0)) and loaded
into a 0.5-ml bed of nickel-nitrilotriacetic acid Superflow (Qiagen)
column pre-equilibrated with binding buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole (pH 8.0)). The column was washed with wash
buffer C (50 mM NaH2PO4, 1 M NaCl, 20 mM imidazole (pH 8.0)), and bound
proteins were eluted with elution buffer B (50 mM
NaH2PO4, 300 mM NaCl, 250 mM imidazole (pH 8.0)). Peak fractions were then pooled and
dialyzed against buffer 88lK (20 mM HEPES (pH 6.8), 75 mM KOAc, 250 mM sorbitol, 5 mM
Mg(OAc)2, 10% glycerol).
-D-galactopyranoside at
37 °C for 3 h and purified using the above mentioned
glutathione-Sepharose column chromatography and MonoQ (Amersham
Biosciences) column chromatography, subsequently. The peak fractions
were then pooled and dialyzed against buffer 88lK.
-32P]ATP (Institute
of Isotopes, Budapest, Hungary). [K+] was adjusted
to 20 mM with KCl in each reaction. After reaction at
25 °C, SDS was added to 1%, and 0.5 µl of the resulting reaction mixture was developed on a polyethyleneimine cellulose thin layer plate
(Sigma) with 0.5 M formic acid, 0.5 M LiCl to
detect released inorganic 32P by BAS imaging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
[in a new window]
Fig. 1.
Amino acid sequence and computational
analysis of JPDI. A, deduced amino acid sequence
of murine JPDI. The dotted underline and arrow,
respectively, indicate the predicted translocation signal sequence and
its cleavage site. The underline and double
underlines, respectively, indicate the J-domain and
thioredoxin-like motifs. A conserved HPD sequence in the J-domain motif
is shadowed, and the His residue replaced by Gln in the H63Q
mutation is indicated in boldface. A putative
N-linked glycosylation site is indicated by an open
circle, and the KDEL motif is boxed. HA-tagged JPDI
(JPDI-HA) carries a HA tag at the position indicated by an
asterisk. B, schematic representation of murine
JPDI. C, comparison of JPDI amino acid sequences in mouse,
human (GenBankTM accession number AK027647), and C. elegans (GenBankTM accession number AL032657).
Identical amino acids are shadowed. The J-domain and
thioredoxin-like motifs are underlined and
double-underlined, respectively.
View larger version (45K):
[in a new window]
Fig. 2.
Northern blot detection of JPDI
mRNA. A, a mouse multi-tissue Northern blot (equal
amount of poly(A)+ RNA blotted in each lane,
Clontech) was hybridized with a JPDI cDNA probe
(top) and a -actin probe (bottom),
respectively. B, NIH3T3 cells were cultured in a
non-stressed condition (no treatment) or under ER-stressed conditions
(3 µg/ml tunicamycin and 0.5 µg/ml thapsigargin, respectively) for
6 h, and the indicated mRNAs were detected by Northern
blotting of the total RNA preparations. JPDI mRNA was detected by
8-fold longer exposure to the BAS imaging plate as compared with BiP
mRNA. Glyceraldehyde3-phosphate dehydrogenase
(GAPDH) mRNA was used as the internal standard.
C, JPDI mRNA shown in panel B was quantified
and normalized against glyceraldehyde-3-phosphate dehydrogenase
mRNA. Normalized mRNA values are presented relative to their
level of expression in untreated cells.
cells (data not shown).
Unlike the result in multi-tissue Northern blot, only the 3.3-kilobase
transcript was detected in these cultured cells.
View larger version (23K):
[in a new window]
Fig. 3.
Subcellular localization of JPDI-HA.
A, NIH3T3 cells were transiently transfected with
pCAG-JPDI-HA and incubated for 36 h. Cells then were fixed and
co-stained with mouse 12CA5 anti-HA and rabbit anti-PDI antibodies.
Fluorescence microscopic images were obtained using fluorescein
isothiocyanate- and rhodamine-conjugated secondary antibodies.
B, 36 h post-transfection of pCAG-JPDI-HA, HeLa cells
were lysed and treated with EndoH for the indicated times, and JPDI-HA
was detected by anti-HA Western blot analysis. g and
d indicate glycosylated and deglycosylated forms,
respectively. A nonspecific protein band is indicated by an
asterisk. C, 36 h post-transfection of
pCAG-JPDI-HA, HeLa cells were homogenized for preparation of
microsomes. The resulting microsomes were incubated with or without
proteinase K (ProK) and Triton X-100 (1% Trx-100) and
subsequently subjected to anti-HA and anti-BiP Western blot analysis. A
nonspecific protein band is indicated by an asterisk.
View larger version (20K):
[in a new window]
Fig. 4.
GST pull-down assay for JPDI J-domain and BiP
binding. A, confirmation of purity of proteins used in
this assay. Purified preparations of protein (350 ng of protein/lane)
were separated by SDS-PAGE (12%) and stained by CBB. B,
glutathione-Sepharose beads carrying the indicated proteins were
incubated with BiP and 0 mM (ATP ) or 1 mM (ATP +) ATP. After washing the beads,
pulled-down proteins were separated by SDS-PAGE (12%) and stained by
CBB.
View larger version (20K):
[in a new window]
Fig. 5.
Assays for ATP hydrolysis by BiP.
A, 1 µg of recombinant BiP (140 nM) was
incubated with 0.5 µg each of GST, GST-J, or GST-J(H63Q) in 100 µl
of ATPase assay buffer at 25 °C as described under "Experimental
Procedures." Samples were collected at indicated times and then
spotted onto a thin layer chromatography for separation. The hydrolysis
of ATP was measured by BAS imaging. B, specific ATPase
activity was calculated from a time-dependent linear
increment of released inorganic 32P in ATP hydrolysis
reactions for 0, 10, 20, 30, 40, 50, and 60 min from A. C, ATP hydrolysis reactions were performed for 40 min at
various concentrations of the indicated proteins. BiP was used at 1 µg (=140 nM) in all reactions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Linda M. Hendershot (St. Jude Children's Research Hospital, Memphis, TN) for a plasmid and Miki Matsumura for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for Scientific Research (to K. K. and A. T.), Encouragement of Young Scientists (Y. K.), and Scientific Research on Priority Areas (K. K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from Novartis Foundation and Yamada Science Foundation.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.
To whom correspondence should be addressed: Research and Education
Center for Genetic Information, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.:
81-743-72-5640; Fax: 81-743-72-5649; E-mail:
kkouno@bs.aist-nara.ac.jp.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M208346200
1
The abbreviations used are: ER,
endoplasmic reticulum; PDI, protein disulfide isomerase; JPDI,
J-domain-containing PDI-like protein; EST,
expressed sequence tag; HA, hemagglutinin; JPDI-HA, HA-tagged JPDI;
EndoH, endo--N-acetylglucosaminidase H; GST, glutathione
S-transferase; GST-J, a fusion of GST-JPDI J-domain (His)6 tag; Trx-100, Triton X-100; CBB, Coomassie Brilliant
Blue R-250; RACE, rapid amplification of cDNA ends.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Stevens, F. J., and Argon, Y. (1999) Semin. Cell Dev. Biol. 10, 443-454[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gething, M.-J. (1999) Semin. Cell Dev. Biol. 10, 465-472[CrossRef][Medline] [Order article via Infotrieve] |
3. | Plemper, R. K., Bohmler, S., Bordallo, J., Sommer, T., and Wolf, D. H. (1997) Nature 388, 891-895[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Brodsky, J. L.,
Werner, E. D.,
Dubas, M. E.,
Goeckeler, J. L.,
Kruse, K. B.,
and McCracken, A. A.
(1999)
J. Biol. Chem.
274,
3453-3460 |
5. |
Nishikawa, S. I.,
Fewell, S. W.,
Kato, Y.,
Brodsky, J. L.,
and Endo, T.
(2001)
J. Cell Biol.
153,
1061-1070 |
6. | Vogel, J. P., Misra, L. M., and Rose, M. D. (1990) J. Cell Biol. 110, 1885-1895[Abstract] |
7. | Hamman, B. D., Hendershot, L. M., and Johnson, A. E. (1998) Cell 92, 747-758[Medline] [Order article via Infotrieve] |
8. | Matlack, K. E., Misselwitz, B., Plath, K., and Rapoport, T. A. (1999) Cell 97, 553-564[Medline] [Order article via Infotrieve] |
9. | Bertolotti, A., Zhang, Y., Hendershot, L. M., Harding, H. P., and Ron, D. (2000) Nat. Cell Biol. 2, 326-332[CrossRef][Medline] [Order article via Infotrieve] |
10. | Okamura, K., Kimata, Y., Higashio, H., Tsuru, A., and Kohno, K. (2000) Biochem. Biophys. Res. Commun. 279, 445-450[CrossRef][Medline] [Order article via Infotrieve] |
11. | Bukau, B., and Horwich, A. L. (1998) Cell 92, 351-366[Medline] [Order article via Infotrieve] |
12. | Kelley, W. L. (1998) Trends Biochem. Sci. 23, 222-227[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Yu, M.,
Haslam, R. H.,
and Haslam, D. B.
(2000)
J. Biol. Chem.
275,
24984-24992 |
14. | Wawrzynow, A., and Zylicz, M. (1997) in Molecular Chaperones and Protein Folding Catalysts (Gething, M.-J., ed) , pp. 95-98, Oxford University Press, Oxford, United Kingdom |
15. |
Silberstein, S.,
Schlenstedt, G.,
Silver, P. A.,
and Gilmore, R.
(1998)
J. Cell Biol.
143,
921-933 |
16. | Brodsky, J. L. (1997) in Molecular Chaperones and Protein Folding Catalysts (Gething, M.-J., ed) , pp. 108-110, Oxford University Press, Oxford, United Kingdom |
17. | Skowronek, M. H., Rotter, M., and Haas, I. G. (1999) Biol. Chem. Hoppe-Seyler 380, 1133-1138 |
18. |
Chevalier, M.,
Rhee, H.,
Elguindi, E. C.,
and Blond, S. Y.
(2000)
J. Biol. Chem.
275,
19620-19627 |
19. |
Dudek, J.,
Volkmer, J.,
Bies, C.,
Guth, S.,
Muller, A.,
Lerner, M.,
Feick, P.,
Schafer, K. H.,
Morgenstern, E.,
Hennessy, F.,
Blatch, G. L.,
Janoscheck, K.,
Heim, N.,
Scholtes, P.,
Frien, M.,
Nastainczyk, W.,
and Zimmermann, R.
(2002)
EMBO J.
21,
2958-2967 |
20. |
Shen, Y.,
Meunier, L.,
and Hendershot, L. M.
(2002)
J. Biol. Chem.
277,
15947-15956 |
21. | Noiva, R. (1999) Semin. Cell Dev. Biol. 10, 481-493[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Freedman, R. B.,
Klappa, P.,
and Ruddock, L. W.
(2002)
EMBO Rep.
3,
136-140 |
23. | Darby, N. J., and Creighton, T. E. (1995) Biochemistry 34, 16770-16780[Medline] [Order article via Infotrieve] |
24. | Tsai, B., Rodighiero, C., Lencer, W. I., and Rapoport, T. A. (2001) Cell 104, 937-948[Medline] [Order article via Infotrieve] |
25. | Munro, S., and Pelham, H. R. (1987) Cell 48, 899-907[Medline] [Order article via Infotrieve] |
26. | Pelham, H. R. (1996) Cell Struct. Funct. 21, 413-419[Medline] [Order article via Infotrieve] |
27. | Kimata, Y., Ooboki, K., Nomura-Furuwatari, C., Hosoda, A., Tsuru, A., and Kohno, K. (2000) Gene (Amst.) 261, 321-327[CrossRef][Medline] [Order article via Infotrieve] |
28. | Kozak, M. (1984) Nucleic Acids Res. 12, 857-872[Abstract] |
29. | Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193-199[CrossRef][Medline] [Order article via Infotrieve] |
30. | Iwawaki, T., Hosoda, A., Okuda, T., Kamigori, Y., Nomura-Furuwatari, C., Kimata, Y., Tsuru, A., and Kohno, K. (2001) Nat. Cell Biol. 3, 158-164[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Wei, J.,
and Hendershot, L. M.
(1995)
J. Biol. Chem.
270,
26670-26676 |
32. |
Corsi, A. K.,
and Schekman, R.
(1997)
J. Cell Biol.
137,
1483-1493 |
33. | Kawai, J., Shinagawa, A., Shibata, K., Yoshino, M., Itoh, M., Ishii, Y., Arakawa, T., Hara, A., Fukunishi, Y., Konno, H., Adachi, J., Fukuda, S., Aizawa, K., Izawa, M., Nishi, K., Kiyosawa, H., Kondo, S., Yamanaka, I., Saito, T., Okazaki, Y., Gojobori, T., Bono, H., Kasukawa, T., Saito, R., Kadota, K., Matsuda, H., Ashburner, M., Batalov, S., Casavant, T., Fleischmann, W., Gaasterland, T., Gissi, C., King, B., Kochiwa, H., Kuehl, P., Lewis, S., Matsuo, Y., Nikaido, I., Pesole, G., Quackenbush, J., Schriml, L. M., Staubli, F., Suzuki, R., Tomita, M., Wagner, L., Washio, T., Sakai, K., Okido, T., Furuno, M., Aono, H., Baldarelli, R., Barsh, G., Blake, J., Boffelli, D., Bojunga, N., Carninci, P., de Bonaldo, M. F., Brownstein, M. J., Bult, C., Fletcher, C., Fujita, M., Gariboldi, M., Gustincich, S., Hill, D., Hofmann, M., Hume, D. A., Kamiya, M., Lee, N. H., Lyons, P., Marchionni, L., Mashima, J., Mazzarelli, J., Mombaerts, P., Nordone, P., Ring, B., Ringwald, M., Rodriguez, I., Sakamoto, N., Sasaki, H., Sato, K., Schonbach, C., Seya, T., Shibata, Y., Storch, K. F., Suzuki, H., Toyo-oka, K., Wang, K. H., Weitz, C., Whittaker, C., Wilming, L., Wynshaw-Boris, A., Yoshida, K., Hasegawa, Y., Kawaji, H., Kohtsuki, S., and Hayashizaki, Y. (2001) Nature 409, 685-690[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Lee, K.,
Tirasophon, W.,
Shen, X.,
Michalak, M.,
Prywes, R.,
Okada, T.,
Yoshida, H.,
Mori, K.,
and Kaufman, R. J.
(2002)
Genes Dev.
16,
452-466 |
35. |
Wall, D.,
Zylicz, M.,
and Georgopoulos, C.
(1994)
J. Biol. Chem.
269,
5446-5451 |
36. |
Wawrzynow, A.,
and Zylicz, M.
(1995)
J. Biol. Chem.
270,
19300-19306 |
37. |
Tsai, J.,
and Douglas, M. G.
(1996)
J. Biol. Chem.
271,
9347-9354 |
38. | Schlenstedt, G., Harris, S., Risse, B., Lill, R., and Silver, P. A. (1995) J. Cell Biol. 129, 979-988[Abstract] |
39. | Lin, H. Y., Masso-Welch, P., Di, Y. P., Cai, J. W., Shen, J. W., and Subjeck, J. R. (1993) Mol. Biol. Cell 4, 1109-1119[Abstract] |
40. |
Mayer, M.,
Kies, U.,
Kammermeier, R.,
and Buchner, J.
(2000)
J. Biol. Chem.
275,
29421-29425 |
41. |
Oliver, J. D.,
van der Wal, F. J.,
Bulleid, N. J.,
and High, S.
(1997)
Science
275,
86-88 |
42. |
Oliver, J. D.,
Roderick, H. L.,
Llewellyn, D. H.,
and High, S.
(1999)
Mol. Biol. Cell
10,
2573-2582 |