In the present study, we isolated and characterized a cDNA clone from Cercopithecus aethiops monkey cells, Mydj2, which is similar to mouse Hsj2 [23].. Cell culture and heat treatment: W
Trang 1Mydj2 as a potent partner of hsc70 in mammalian cells
Petros Bozidis, Ioannis Lazaridis, Gerassimos N Pagoulatos and Charalampos E Angelidis
Laboratory of General Biology, Medical School, University of Ioannina, Greece
Dj2 is a member of the DnaJ family of proteins, which
regulate the chaperoning function of the hsp70s We isolated
a monkey cDNA dj2 clone corresponding to the large
mRNA species encoded by the gene This mRNA differs
from the small mRNA produced by the same gene in that it
contains a long 3¢ untranslated region Both messages were
found to be equally stable and to produce the same protein,
which is susceptible to farnesylation Studies in mouse tissues
and various cell lines revealed that these messages and their
products are differentially expressed Surprisingly, we found that only the nonfarnesylated form of dj2 is capable of translocating to the cell nucleus, especially after heat shock Finally, based on protein interaction studies, our results indicate that dj2 is a specific partner for hsc70 and not for hsp70
Keywords: DnaJ homologue; dj2; heat shock; cochaperone; nanomachine
It is widely accepted today that the eukaryotic DnaJ
homologs consist a family of proteins which in combination
with the hsp70 family members make up the basic molecular
chaperone machinery of the mammalian cell [1] The
members of the above gene families work together in a
variety of cellular processes, including protein folding during
which the hsp70s bind unfolded, partially folded or
dena-tured polypeptide substrates and assist their renaturation
through a cycle of binding and release regulated by their
DnaJ cochaperones [2,3] Based on the existense of three
dinstict domains, namely: the highly conserved J domain
consisting of approximately 70 amino acids and known to
mediate the hsp70 binding; the glycine/phenylalanine (G/F)
rich region, which possibly acts as a flexible linker; and the
cysteine rich region (C domain) which resembles a
zing-finger domain, a large number of eukaryotic DnaJs have
been identified and classified accordingly [4]
In the cytosol of human cells, four DnaJ homologs have
been identified, namely: dj1 or hsp40/hdj1 [5–7], dj2 or
HSDJ/hdj2 [7,8], hsj1 [9] and dj3 or DNJ3/rdj2 [10] Among
them dj2 has the closest structural similarity to the bacterial
DnaJ, as it contains all three characteristic domains, that is,
the J domain, the G/F domain and the cysteine rich region
Furthermore, dj2 contains a C-terminal ÔCaax boxÕ
prenyl-ation motif common to proteins which are
post-transla-tionally farnesylated [11] The zinc finger domain seems to
be important for the binding with chemically denatured
luciferase [12] Although it is still not clear how the G/F
domain modulates the interaction of the J domain with
hsp70, it has been proposed recently that this domain is
responsible for stimulating the J domain function [12,13]
However in all DnaJ proteins, even in the absence of a G/F
domain, the presence of the J domain is sufficient to mediate
some form of hsp70 regulation [14] Finally the post-translational farnesylation is considered to be an important process, because it seems to facilitate the chaperoning function [15] or the binding to membranes [16]
Dj2 was found to cooperate with hsc70 in assisting the folding of denatured proteins [10,17] and in participating in the process of protein import into the mitochondria and the endoplasmic reticulum [18,19] It was also shown that dj2 facilitates the early steps of transmembrane receptor biogenesis in cystic fibrosis [20] and is mobilized to the nucleus in order to refold misfolded receptors into biolo-gically active conformation states [17] Finally, overexpres-sion of dj2 was recently found to decrease aggregate formation caused by expanded polyglutamine tracts, a hallmark of neurodegenerative diseases [21,22]
In the present study, we isolated and characterized a cDNA clone from Cercopithecus aethiops (monkey) cells, Mydj2, which is similar to mouse Hsj2 [23] This cDNA corresponds to an ortholog of the DNAJA1 gene according
to the nomenclature suggested by Ohtsuka & Hata [24] it should be named caDjA1 Comparison of Mydj2 and Hsj2 with the HDJ2 [7] and hdj2 [8] cDNAs, showed that our clone although similar to Hsj2, has an extended 3¢ noncod-ing region of 981 bp To determine whether the additional sequences influence the stability of the RNA, as previously reported for other RNAs [25] studies addressing this question were performed and showed that there is no difference in the stability of the two dj2 mRNAs Further-more, we investigated the in vivo properties of the endo-genous Mydj2 in mammalian cells We found that only the nonfarnesylated form of dj2 translocates to the nucleus especially after heat shock and that dj2 binds only to the constitutive form of hsp70, namely hsc70
M A T E R I A L S A N D M E T H O D S
CDNA library screening
A cDNA library, prepared using RNA isolated from COS cells was obtained from Stratgene (monkey COS cell line cDNA k ZAPRII, Cat no 936110) The library was screened using the entire hudj2 cDNA as a probe Library
Correspondence to A E Charalampos, University of Ioannina,
Medical School, Laboratory of General Biology, Ioannina, 45110.
Fax: + 30 0651 97863, Tel.: + 30 0651 97567,
E-mail: chaggeli@cc.uoi.gr
Abbreviations: HSP, heat shock protein; My, monkey; Hu, human.
(Received 11 October 2001, revised 18 January 2002, accepted
23 January 2002)
Trang 2plating, phage DNA lifts, hybridization and washes,
isolation of positive clones, and excision of phagemids were
performed according to Stratagene’s instructions
Animals
Adults F1 male mice [26] were sacrificed under chloroform/
ether (1 : 1, v/v) atmosphere and organs or tissues were
excised and immediately placed in ice cold NaCl/Pi After
repeated washes with cold NaCl/Pi the organs or tissues
were elaborated for RNA preparation and Northern blot
analysis or electrophoresis and Western blotting or frozen in
liquid nitrogen and stocked at)180 °C for further use
Cell culture and heat treatment: Western
blotting of cell and tissues lysates
Monkey kidney CV1 cells were grown in monolayers as
described previously [27] Subconfluent cells were
heat-treated by immersing the culture dishes in a water bath set at
the desired temperature
Sub-confluent control or heat treated cells were
harves-ted, washed with NaCl/Piand resuspended in 300 lL RIPA
buffer (50 mMTris/HCL, 150 mMNaCl, 1% Triton X-100,
1% sodium deoxycholate, 0.1% SDS) with 1 lgÆmL)1
pepstatin, 1 lgÆmL)1leupeptin, 1 mM
phenylmethanesulfo-nyl fluoride and 10 UÆmL)1apyrase Lysates were prepared
after incubation of the cell suspension on ice for 10 min
During this period the lysates were homogenized by passing
five or six times through a 21-guage needle, followed by
centrifugation at 12 900 g for 10 min at 4°C (Eppendorf)
The supernatants were mixed with SDS-sample buffer at
final concentration (62.5 mM Tris/HCl, pH 6.8, 5%
2-mercaptoethanol, 3% SDS, 10% glycerol, 0.1%
bromo-phenol blue), boiled for 3 min and used to electrophoresis in
10% polyacrylamide/SDS gels and Western-blot analysis
using the enhanced chemiluminescence method (ECL,
Amersham International, Amersham, Bucks, UK)
After extensive washings with cold NaCl/Pithe organs or
the tissues were homogenized in SDS/lysis buffer (100 mM
Tris/HCL, pH 8.7, 2% SDS, 5% 2-mercaptoethanol and
15% glycerol) The resulting samples were heat-denatured
at 100°C for 3 min and then sonicated at 50-W for 5 s, to
shear the DNA [28] The suspensions were finally mixed
with SDS sample buffer and processed as above
Approximately 30 lg of proteins were analyzed by 10%
SDS/PAGE mini-gel and processed by Western blotting
[29]
Plasmid constructs
The full length of human cDNA dj2 clone [8] corresponding
to the small dj2 mRNA species, was subcloned to pBL-KS
plasmid at SalI/NotI sites
The clone 5aI cDNA corresponding to the large dj2
mRNA species, was subcloned to pBL-SK plasmid at
EcoRI site
In order to bacterially express Mydj2 the corresponding
cDNA was amplified by PCR using primer I (5¢-GCA
GTAGAGGATCCTGAAAGAAA-3¢) and primer II
(5¢-GTTATTCAGTCGACCATTAAGAGG-3¢) to
gener-ate the convenient BamHI (at 5¢ end) and SalI (at 3¢ end)
restriction sites The amplified product was then ligated, in
frame with 6·His, into the BamHI and SalI sites of the pQE-32 plasmid (Qiagen, GmbH, Germany) resulting in the generation of the pQE-32-Mydj2 plasmid The accuracy
of the resulting construct (pQE-32-Mydj2) was verified by DNA sequencing, and the plasmid was subsequently used to overexpress Mydj2–6·His in Escherichia coli
Expression and purification of histidine-tagged Mydj2: antibody production
The pQE-32-Mydj2 plasmid was used to overexpress the Mydj2 protein Overnight cultures of E coli JM109 carry-ing pQE-32-Mdj2 plasmid were diluted 10-fold and cultured for 1 h After isopropyl thio-b-D-galactoside induction (2 mM) for 2 h at 37°C, the cells were collected by brief centrifugation and cell lysates were prepared by sonication The recombinant protein was purified from the cell lysates using a Ni/nitrilotriacetic acid column and imidazole elution (50–250 mM) as described by the manufacturers (Qiagen) Anti-Mydj2 antibodies were obtained by injecting a male rabbit with purified Mydj2 protein [30]
Protein–protein interactions experiments For immunoprecipitations, cell lysates prepared in RIPA buffer were incubated overnight at 4°C by end-over-end rocking with 5 lL of hsc70-specific antibody (SPA-815, StressGene), 3 lL hdj2-specific antibody, or 3 lL anti-hsp70 Ig (Amersham, RPN 1197) Protein [A-G]–Sepharose beads (Promega: cat no sc-2003) were then added to the reaction and incubation was continued for an additional
60 min The immunoprecipitates were collected by centri-fugation, washed three times with RIPA buffer, mixed with SDS sample buffer at 1· final concentration, boiled for
5 min and subjected to SDS/PAGE and Western-blot analysis
Pull-down experiments using 6·Hiss-Mydj2 immobilized
on Ni/nitrilotriacetic acid resin were performed as previ-ously described [31,32] except for minor modifications More specifically purified 6·His-Mydj2 protein was immo-bilized and refolded on Ni/nitrilotriacetic acid resin accord-ing to the manufacturer’s instructions (Qiagen) RIPA cell lysates from 2.5· 106CV1 or COS cells were prepared as described above, mixed with approximately 2 lg dj2-His purified protein immobilized on Ni/nitrilotriacetic acid resin (Qiagen) and incubated at 4°C by end-over-end rocking for
2 h Proteins bound to the dj2-Ni/nitrilotriacetic acid resin were precipitated by centrifugation at 664 g for 5 min at
4°C, washed extensively (three times) with RIPA, mixed with SDS-sample buffer to 1· final concentration, boiled for 5 min and analyzed by 10% SDS-mini PAGE and Western blotting
Indirect immunofluorence CV1 cells growing in coverslips were incubated at 37°C or
43°C for 2 h as indicated in the text and figure legends They were then washed twice with cold NaCl/Piand fixed for 10 min, at room temperature in 2% paraformaldehyde The cells were washed three times with cold NaCl/Pi and permeabilized by incubating in ice cold, absolute methanol for 3–5 min at 20°C Then, the cells were washed three times with cold NaCl/Pand incubated in 3% BSA in
Trang 3NaCl/Pi to prevent nonspecific staining After 1 h of
incubation with blocking medium, the cells were washed
three times with NaCl/Pi and incubated for 1 h with
20–30 lL of the primary antibody diluted as indicated in
NaCl/Pi/3% BSA Following washings (three times),
20–30 lL of the secondary antibody, fluorescein
isothio-cyante-conjugated goat anti-(rabbit IgG) Ig diluted 1 : 25 in
NaCl/Pi, was added After 1 h of incubation the cells on the
coverslip were washed three times with NaCl/Pi, placed at
the opposite site on a glass-slide with a drop of glycerol and
observed in an immunofluorence microscope
RNA methods
Total RNA from mammalian cells and mouse tissues, was
isolated as described previously [33]
Total RNA (10–20 lg) were electrophoresed in a
for-maldehyde-containing 1% agarose gel and transferred to a
nylon membrane (Amersham: HybondTM-N, code RPN
303 N) The membrane was hybridized with the 612-bp
large dj2 fragment or with the entire Mydj2 cDNA labeled
with [a-32P] dCTP (NEN: 3000 CiÆmmol)1, NEG 513H) as
indicated, washed, and exposed to Kodak XAR film at
)70 °C for 2–5 days using Kodak lightening plus screens
RNA stability assays were carried out as described [25]
Actinomycin D (Sigma: 10 mgÆmL)1in dimethylsulfoxide)
was added to COS and HeLa cells to a final concentration
of 10 lgÆmL)1 After 0, 1, 2, 3 and 4 h of incubation with
actinomycin D, cells (8· 106) were washed with NaCl/Pi
and total RNA was prepared Northern blot analysis and
detection of both messages, large dj2 and small dj2 mRNAs,
were performed as described previously [34]
R E S U L T S
Isolation of a cDNA clone encoding for Mydj2
Using the methodology described above we isolated a
2.3-kb full length monkey cDNA which encodes a DnaJ
homologue (GenBank accession number: AF395203) and
more specifically the ortholog of the human dj2 protein As
a cDNA source we used the premade by Stratagene cDNA
library of monkey COS cells which was screened using the
entire human dj2 cDNA clone [8] Among several positive
clones, one clone (5aI) with a 2.2-kb insert was isolated
Nucleotide sequence analysis revealed that 5aI clone had a
single open reading frame of 397 amino-acids beginning
with the ATG codon at nucleotide 36–38 and terminating
with the TAG codon at nucleotide 1228–1230 Comparison
of our clone (5aI) with the human dj2 cDNA [7,8] showed
that clone 5aI stems from a larger mRNA having 0.9-kb
additional sequences at the 3¢ UTR (Fig 1A)
Further sequence comparison between the isolated
mon-key dj2 cDNA (clone 5aI) and the human dj2 cDNA
showed an identity of 99% for the J domain, 100% for the
G/F region, 99% for the cysteine rich domain (cysteine rich
region) and 97% for the C-terminus (Fig 1A)
Differential expression of large and small dj2 mRNAs
The distribution of dj2 RNAs in different cell lines was
initially studied We used as DNA probes the entire coding
region of the Mydj2 gene (Fig 1A), which can hybridize to
both dj2 mRNA species (Fig 1B, left panel), or the fragment between 1517 and 2129 bp, in the 3¢ UTR region, which can hybridize only to the large dj2 mRNA (Fig 1B, right panel) Following our theoretical approach we examined the expression of both dj2 RNAs in different mammalian cells and mouse tissue extracts in order to clarify the possibility of preferential expression RNAs from COS, HeLa, F9 and U937 cells were used in Northern blot analysis The small mRNA was found to be abundantly expressed in all cell types and only in F9 cells both messages were detected at very low levels (Fig 1B, left panel) The large dj2 mRNA, compared to the small dj2 mRNA, was found to be at least three to five times lower (Fig 1B, left panel) and this ratio did not change when the cells were exposed to heat shock (Fig 1B) When the 612 bp fragment of the 3¢ UTR region (1517–2129 bp) was used as a probe, only the large dj2 mRNA was identified (Fig 1B, right panel)
To analyse the tissue distribution of large and small dj2 mRNAs, we isolated RNAs from a number of mouse tissues (Fig 2A) Northern blot analysis, using the entire Mydj2 cDNA as a probe, revealed that both messages can
be detected in all tissues but their distribution is quite different Small dj2 mRNA was found to represent the major member of the two message population (Fig 2A), which was abundant in all tissues examined except skeletal muscle In contrast large dj2 mRNA was found to be abundant mainly in the brain, kidney and lung (Fig 2A) The major observation of this study was that the large dj2 mRNA distribution differed from that of the small dj2 and that the levels of both messages varied according to the cell
or tissue type The significanse of the above finding remains
to be clarified
We then examined the distribution of the corresponding dj2 protein in the same rat and mice tissues (Fig 2B,C,D) For this study we used two dj2 specific antibodies, one which
Fig 1 Characterization and comparison of large and small dj2 mRNAs (A) Schematic representation and sequence comparison of the monkey large dj2 cDNA and human small dj2 cDNA (B) Northern blot analysis of the two forms of dj2 mRNAs, using RNAs from control (–)
or heat treated for 90 min at 43 °C and 90 min recovery to 37 °C (+) monkey COS, human HeLa, mouse teratocarcinoma F9 and human histocytic lymphoma U-937 cell lines The position of 18S rRNAs is shown at the bottom.
Trang 4recognizes the entire monkey dj2 protein (see Materials and
methods) and another that recognizes only the N-terminal
end (1–179 amino acids) of the human dj2 protein
(Neomarkers, cat no MS225P) Samples from rat (Fig 2B)
and mouse tissues (Fig 2C,D) contained the same amounts
of protein were used for this study Dj2 protein levels were
found to be particularly high in testis, brain, kidney and
liver On the other hand, tissues like the heart, muscle and
lung revealed lower but detectable amounts of dj2 proteins
(Fig 2B,C,D) Unexpectedly, under our experimental
con-ditions, the identification of the farnesylated and the
nonfarnesylated dj2 forms was not possible regardless of
the type of antibodies and tissues used However, one
particular feature of dj2 expression was the possible
existence of farnesylated forms or isoforms of dj2 in testis,
which were identified only with our anti-dj2 Ig (Fig 2B,C)
Large and small dj2 mRNAs are stable
and their degradation rates are similar
To further investigate the role of the extensive 3¢ UTR in
our clone (5aI) we addressed the possibility that this
structure may play a significant part in the regulation and stability of the message, given that instability elements were found to exist in the 3¢ UTR [35] Therefore COS and HeLa cells were treated with Actinomycin D for different periods
of time After the incubated periods, RNAs were prepared and the samples were subjected to Northern blotting analysis, using the full length Mydj2 cDNA as a probe Detection of the large and small Mydj2 mRNAs under the conditions described revealed that both messages were very stable Even 24 h after the treatment with actinomycin D, both messages were present in detectable quantities (Fig 3A) It should also be noted that this mRNA stability does not change under the same conditions and exposure of cells to heat shock of 43°C for 90 min (Fig 3B)
Only the nonfarnesylated form of dj2 translocates
to the nucleus
In order to confirm the open reading frame of the isolated Mydj2 cDNA (clone 5aI) and detect the in vitro products that our clone is able to produce, the dj2 (5aI) cDNA was cloned in the T7/T3 expression vector pBL-KS The PCR product of Hudj2 coding region was also cloned in the same vector while the full length, with 5¢ UTR and the small 3¢ UTR, of Mydj2 cDNA (clone 5aI) was cloned in the pBL-SK expression vector In vitro transcription/translation
of the above subcloned DNAs revealed that all of them were able to produce the dj2 protein with a molecular mass of approximately 46–53 kDa, as expected (data not shown) The dj2 protein produced was found to be susceptible to farnesylation and the inhibition of farnesylation in CV1
Fig 2 Differential expression of dj2 in mouse and rat tissues (A) RNA
blot analysis was performed for dj2 in mouse tissues Total RNAs
(20 lg) from lung, brain, testis, kidney, heart, spleen, muscle, liver and
from COS and HeLa cells, were analyzed by Northern blotting, using
as probe the 32 P-labeled cDNA for monkey dj2 The position of 18S
rRNAs is shown at the bottom (B,C,D) Dj2 protein distribution in
tissues of Wistar rats (B) and mice (C,D) Tissue total cell extracts were
obtained as described in Materials and methods Equal amounts of
proteins were analyzed by immunoblotting using specific antibodies
for the entire dj2 protein (B,C) and for the N-terminal fragment of dj2
(D) P, denotes the purified recombinant Mydj2 protein The lower
band observed in testis probably represents a testis specific dj2
ortholog or a modified form of dj2.
Fig 3 Both large and small dj2 mRNAs are stable molecules COS and HeLa cells were treated with 10 lgÆmL)1actinomycin-D for 0, 2, 4, 16 and 24 h (A) Parallel cultures were treated in the same way, with actinomycin D and exposed in heat shock for 90 min at 43 °C (B) Then, RNAs were prepared and 20 lg of each sample were subjected to RNA blot analysis using the entire Mydj2 cDNA, radio-labelled with [c32P]dCTP, as a probe Integrity of RNAs was verified
by the apparently identical intensities of 18S rRNAs.
Trang 5cells, using a-hydroxy-farnesyl phosphonic acid, showed
that Mydj2, in agreement with previous studies [10,30] exists
in two forms, a farnesylated and a nonfarnesylated one
(data not shown)
We next examined the intracellular localization pattern of
dj2, under physiological or heat shock conditions (43°C for
90 min and 60 min recovery at 37°C), utilizing our anti-dj2
Ig, which was raised against the entire Mydj2 molecule, in
immunofluorescence experiments As shown in Fig 4B, dj2
is diffused within the cell but significant amounts of the
protein can be detected mainly in the cytoplasm However a
larger quantity of the protein seems to accumulate in the
nucleus and especially in the nucleolus after heat shock
(Fig 4) In order to further investigate the above
phenom-enon, we proceeded in examining the intracellular
localiza-tion of both dj2 forms (farnesylated and nonfarnesylated) using subcellular fractionation techniques [27]
Whole cell or cytoplasmic and nuclear extracts from control and heat-treated CV1 cells were used in order to determine the subcellular localization of the farnesylated and the nonfarnesylated forms of Mydj2 protein by Western blotting analysis, using the same anti-dj2 Ig as in immuno-fluorence experiments As shown in Fig 5, Mydj2 does not seem to be heat-inducible, in CV1 cells at the times and temperatures analyzed, but from its two observed forms only the nonfarnesylated form was found to be translocated
to the nucleus It is also noteworthy that an extra population
of the nonfarnesylated dj2 molecules seems to accumulate to the nucleus after heat shock (Fig 5)
Therefore, we concluded, that under our experimental conditions, dj2 is diffused in the entire cell and only the nonfarnesylated form is translocated to the nucleus Association of cochaperones Mydj2 with Myhsc70 The partner selectivity between chaperones and cochaper-ones is not entirely clear To further define how hsc70 and DnaJ-like proteins interact, we decided to use two meth-odological approaches, one involving immunoprecipitations and another involving a modified pull-down assay CV1 or COS cells were exposed at 42.5°C for 90 min and recovered at 37°C for 90 min RIPA cell extracts from control or heat-shocked cells were then prepared and used in pull-down experiments More specifically, the lysate from 2.5· 106cells was mixed with Mydj2-His purified recom-binant protein immobilized on Ni-nitrilotriacetic acid resin (Qiagen) After incubation, extended washings and centri-fugation of the coprecipitated proteins, all fractions were subjected to SDS/PAGE and Western blotting analysis As shown in Fig 6, hsc70 was found to bind to immobilized Mydj2-His protein (lanes 3, 3¢) In contrast, no binding between the immobilized dj2 protein and the inducible hsp70 protein was observed (Fig 6, lanes: 3, 3¢) Interestingly, the same results were obtained when lysates from heat shocked cells were used (Fig 6, lanes: 6, 6¢) despite the fact that in this case the levels of hsp70 were substantially elevated (compare lanes 1 with 4 and 1¢ with 4¢ in Fig 6) as it was expected
Fig 5 The farnesylated dj2 form translocates to the cell nucleus CV1 cells growing under physiological conditions (37 °C) or heat treated for
120 min at 43 °C followed by recovery at 37 °C for 160 min, were collected and fractionated into whole cell, cytoplasmic and nuclear extracts as described in Materials and methods Control and heat shocked extracts were then subjected to Western blotting analysis utilizing an anti-dj2 Ig P, denotes the purified recombinant Mydj2 protein.
Fig 4 Intracellular distribution of Mydj2 in CV1 cells Cells growing at
37 °C (B) or treated at 43 °C for 120 min followed by recovery at
37 °C for 160 min (C), were fixed and processed for immunofluorence
staining (B) and (C), represent cells stained with anti-dj2 Ig (A)
rep-resents cells stained with preimmune serum.
Trang 6Having shown a specific binding between Myhsc70 and
the recombinant Mydj2-His immobilized on
Ni/nitrilotri-acetic acid agarose beads, we decided to further investigate
these interactions under native or semi in vivo conditions
For that, CV1 cells were harvested and RIPA cell extracts
from control and heat-shocked cells were prepared These
extracts were immunoprecipitated with dj2 or
anti-hsp70 or anti-hsc70 specific Ig as described in Materials and
methods The immunoprecipitated samples were then
resolved by SDS/PAGE and subjected to Western blotting
using the appropriate antibodies As shown in Fig 7, when
an anti-dj2 Ig was used for immunoprecipitation, only the
hsc70 was found to coprecipitate and not the hsp70
(Fig 7C,D) The same pair of proteins was identified to
interact when an anti-hsc70 Ig was used for
immunopre-cipitation (Fig 7A) In contrast, immunopreimmunopre-cipitation with
an anti-hsp70 specific Ig revealed that dj2 does not associate
with the hsp70 protein (Fig 7B)
The above results clearly demonstrate that Mydj2 binds
specifically to hsc70 and that this binding is not susceptible
to changes under elevated temperatures Because the Mydj2
is associated only with the constitutive Myhsc70 we suggest
that these proteins constitute possible partners in the
construction of a cellular chaperoning functional unit
referred to as a chaperoning nanomachine
D I S C U S S I O N
It is known that members of the different chaperone families
are interweaved or combined in order to organize
nanoma-chines For example, the hsp70 family requires cofactors for
specifying its functions A major group of these partners
belong to the DnaJ family [5,8] Little is known about the
specific combination and regulation of all these
nanoma-chines However, we know that chaperones play an essential
role in various cellular functions, such as the acquisition of
thermotolerance and cell survival [26,36,37], the protection
from ischemic injury [38] and in various human disorders
[39–43]
The data presented in this report describe the features of a member of the 40-kDa hsp family, the monkey dj2 protein This member has all the appropriate domains that classify it
as a member of the orthodox DnaJ subfamily The isolated clone 5aI gives an open reading frame of 1191 bp, which is able to produce a polypeptide of 397 amino acids Com-paring our clone with the reported human dj2 [7,8] and mouse dj2 [23] clones, clone 5aI appeared to be similar to the mouse dj2 clone Using clone 5aI as a probe and RNAs from cell lines, we identified two different in size mRNAs The steady-state levels of both messages were examined in different cell lines and mouse tissues In all cases, the ratio of large to small dj2 mRNA ranged between 1 : 2 and 1 : 4 Furthermore, the levels of both messages varied in all cells and tissues examined
Our study showed that the long 3¢ UTR did not contribute to the message stability under normal or heat shocked conditions, given that no known sequences that were responsible of regulating the message stability were found This was in agreement with previous studies, according to which the RNA stability was regulated by ATTTA instability elements [35]
Previous studies have shown that dj2 is mainly present in the microsomal and cytosolic fractions and is translocated to the nucleus [10] or to Golgi, nucleolus and nuclear membrane during heat shock [30] However there is no report indicating which of the two dj2 forms possesses the above properties
Fig 7 The monkey-dj2 is a potential partner to monkey-hsc70 RIPA cell extracts were prepared from control or heat treated at 43 °C for
90 min with 60 min recovery at 37 °C CV1 cells and immunoprecipi-tated with specific antibodies against Mydj2 (C,D), hsc70 (A) and hsp70 (B) The immunoprecipitates were then subjected to Western blotting analysis using Mydj2 (A and B), hsc70 (C) and hsp70 (D) specific antibodies Lane 1, control cell lysate; lane 2, heat shocked cell lysate; lane 4, immunoprecipitate of control lysate; lane 5, immuno-precipitate of heat shocked lysate Lane 3 and 6 represent mock immunoprecipitations of control and heat shocked cell lysates without the corresponding antibodies.
Fig 6 In vitro protein–protein interactions experiment RIPA cell
extracts from control or heat-shocked cells were submitted to a
pull-down assay using His-dj2 on Ni/nitrilotriacetic acid agarose beads as
the binding substrate Cell extracts, washes and eluted protein samples
were analyzed by Western blotting using specific antibodies against
hsp70, hsc70 and Mydj2 1,4,1¢,4¢: cell extracts, 2,5,2¢,5¢: third
wash-ings, 3,6,3¢,6¢: eluted proteins.
Trang 7According to our findings only the nonfarnesylated form of
dj2 is able to translocate into the nucleus In contrast, the
farnesylated form remains localized to the cytosolic fraction
Moreover during heat shock (90 min at 43°C and 60 min
recovery to 37°C), the farnesylated dj2 protein translocates
mostly to the nucleus suggesting that this migration is related
to the facilitation of the folding of the heat denatured nuclear
proteins This result is in agreement with previous
observa-tion which suggests that the HDJ2 protein is mobilized to the
nucleus in response to the presence of inappropriate folded
mutated receptors [17]
Recent studies revealed that hsc70 and dj2 constitute a
potent chaperone pair that is required for mitochondrial
import of preornithine transcarbamylase and refolding of
denatured luciferase [10] or unfolded mutated receptor [17]
In order to verify, in vitro and semi in vivo, the existence of
this functional pair, we performed pull-down and
immu-noprecipitation experiments In pull-down assays using
recombinant Mydj2 fused to 6·His and immobilized to
Ni-nitrilotriacetic acid agarose beads, the binding of
Myhsc70 with Mydj2 was obtained Under the same
conditions hsp70 did not coprecipitate with dj2, which
means that only hsc70 and dj2 can be combined to form a
functional pair
Having identified the Myhsc70 as the direct interaction
partner for Mydj2 by in vitro pull-down experiments, we
tried to repeat the same experiments under semi in vivo
conditions Indeed, we once again identified the existence of
hsc70/dj2 complexes in cell extracts by
coimmunoprecipita-tion of hsc70 with a dj2-specific antiserum or reversibly the
dj2 with a hsc70-specific antiserum We also confirmed that
hsp70 was not able to bind to dj2, indicating that hsc70 and
dj2 constitute potent partners in the construction of a
functional chaperone pair
A C K N O W L E D G E M E N T S
We thank Dr S Kato for the gift of the human dj2 cDNA clone, Dr
Vezyraki for her special contribution on animal handling and making
the anti-dj2 Ig and S Tzialas for his excellent technical contribution.
This work was supported by grants from the Hellenic Ministry of
Research and Technology (PENED-99, # 500) It was also supported
by Empeirikio Institution (11-7-2000) and partially from an EU grant
(QLRT-1999, #30720).
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