Báo cáo Y học: Mydj2 as a potent partner of hsc70 in mammalian cells doc

Mydj2 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 m onkey c DNA d j2 clon e c orresponding to the large
mRNA species encoded by t he gene. T his mRNA differs
from the small mRNA produced by the same gene i n that it
contains a l ong 3¢ untranslated region. Both messages were
found to be equally stable and t o produce the same protein,
which is s usceptible t o f arnesylation. 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 h eat shock.
Finally, b ased on protein interaction studies, our results
indicate that dj2 is a specific partner for hsc70 and not
for h sp70.
Keywords: DnaJ homologue; dj2; h eat s hock; cochaperone;
nanomachine.
It is widely accepted today that the eukaryotic DnaJ
homologs consist a family of proteins which in combination
withthehsp70familymembersmakeupthebasicmolecular
chaperone machinery of the mammalian cell [1]. The
members of the above g ene families work together in a
variety of cellular p rocesses, including p rotein folding during
which the hsp70s bind unfolded, partially folded or dena-
tured polypep tide substrates and assist their renaturation
through a cycle o f binding and release regulated b y 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 a nd known to
mediate the hsp70 binding; the glycin e/phenylalanine (G/F)
rich region, which p ossibly 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 c ytosol 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 i t contains all three characteristic domains, t hat is,
the J domain, the G/F domain and the c ysteine rich region.
Furthermore, dj2 contains a C-terminal ÔCaax boxÕ prenyl-
ation motif common to proteins which are post-transla-
tionally farnesylated [11]. T he zinc finger domain seems to
be impo rtant 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 t hat this domain is
responsible for stimulating the J domain function [12,13].
However in a ll 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]. F inally the post-
translational f arnesylation 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 i n 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 m isfolded 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, whic h 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 w hether 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 t he two d j2 mRNAs. Further-
more, w e investigated the in vivo properties of the endo-
genous Mydj2 in mammalian cells. We found that only the
nonfarnesylated form of d j2 translocates to the nu cleus
especially after heat shock and that dj2 binds only to the
constitutive form of hsp70, namely hsc70.
MATERIALS AND METHODS
CDNA library screening
A cDNA library, prepared using RNA isolated from COS
cells was obtained from Stratgene (monkey COS cell line
cDNA k ZAP

R
II, 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: + 3 0 0651 97863, Tel.: + 30 0651 97567,
E-mail:
Abbreviations: HSP, heat shock protein; My, monkey; Hu, human.
(Received 1 1 October 2001, revised 18 January 2002, accepted
23 January 2002)
Eur. J. Biochem. 269, 1553–1560 (2002) Ó FEBS 2002
plating, phage DNA lifts, hybridization and w ashes,
isolation of positive clones, and excision of phagemids were
performed according to Stratagene’s instructions.
Animals
Adults F1 male mice [26] were sacrificed under c hloroform/
ether (1 : 1, v/v) atmosphere and organs or tissues were
excised and immediately placed in ice c old NaCl/P
i
.After
repeated washes w ith cold NaCl/P
i
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]. S ubconfluent 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/P
i
and resuspended in 300 lLRIPA
buffer (50 m
M
Tris/HCL, 150 m
M
NaCl, 1% Triton X-100,
1% sodium deoxycholate, 0.1% SDS) with 1 lgÆmL
)1
pepstatin, 1 lgÆmL
)1
leupeptin, 1 m
M
phenylmethanesulfo-
nyl fluoride and 10 UÆmL
)1
apyrase. Lysates were prepared
after incubation of the cell s uspension on ice for 1 0 m in.
During this period the lysates were homogenized by passing
five or six times t hrough a 21-guage needle, f ollowed 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 m
M
Tris/HCl, pH 6.8, 5%
2-mercaptoethanol, 3% S DS, 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, B ucks, UK).
After extensive washings with cold NaCl/P
i
the organs or
the t issues were ho mogenized i n SDS/lysis buffer (100 m
M
Tris/HCL, pH 8.7, 2% SDS, 5% 2-mercaptoethanol and
15% glycerol). The resulting samples were he at-denatured
at 100 °C for 3 m in and then sonicated at 5 0-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 t o pBL-KS
plasmid at SalI/NotIsites.
The clone 5aI cDNA corresponding to the large dj2
mRNA species, was subcloned t o pBL-SK plasmid at
EcoRI s ite .
In order to bacterially express Mydj2 the corresponding
cDNAwasamplifiedbyPCRusingprimerI(5¢-GCA
GTAGAGGATCCTGAAAGAAA-3¢) and primer II
(5¢-GTTATTCAGTCGACCATTAAGAGG-3¢) to gener-
ate the convenient BamHI (at 5¢ end) and SalI(at3¢ end)
restriction sites. The amplified product was then ligated, in

frame with 6 ·His, into the BamHI and SalIsitesofthe
pQE-32 plasmid (Qiagen, GmbH, Germany) resulting in
the generation of the pQE-32-Mydj2 plasmid. The accuracy
of the resu lting construct ( pQE-32-Mydj2) was v erified by
DNA sequencing, and the plasmid was subsequently used to
overexpress M ydj2–6·His in Escherichia coli.
Expression and purification of histidine-tagged Mydj2:
antibody production
The p QE-32-Mydj2 plasmid was used to overexpress the
Mydj2 protein. Overnight cultures of E. co li JM109 carry-
ing pQE-32-Mdj2 plasmid were diluted 10-fold and cultured
for 1 h. After isopropyl thio-b-
D
-galactoside induction
(2 m
M
)for2hat37°C, the cells were collected by brief
centrifugation and cell lysates were prepared by sonication.
The recombinant prote in was purified from the cell lysates
using a Ni/nitrilotriacetic acid column and imidazole elution
(50–250 m
M
) 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 prep ared 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, o r 3 lLanti-

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 e xperiments using 6·Hiss-Mydj2 immobilized
on Ni/nitrilotriacetic acid resin were performed as previ-
ously described [31,32] except f or minor modifications.
More specifically purified 6·His-Mydj2 protein was immo-
bilized and refolded o n N i/nitrilotriacetic acid resin accord-
ing to the manufacturer’s i nstructions (Qiagen). RIPA cell
lysates f rom 2 .5 · 10
6
CV1 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 bou nd 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 °Cor
43 °C for 2 h as indicated i n the text and figure legends.
They were then washe d twice with cold NaCl/P

i
and fi xed
for 1 0 m in, at room temperature i n 2% p araformaldehyde.
The cells were washed three times with cold NaCl/P
i
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/P
i
and i ncubated i n 3% BSA in
1554 P. Bozidis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
NaCl/P
i
to prevent nonspecific staining. After 1 h of
incubation with blocking medium, the cells were washed
three times with NaCl/P
i
and incubated for 1 h with
20–30 lL of the primary antibody diluted as indicated in
NaCl/P
i
/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/P
i
, was added. After 1 h of incubation the cells on the
coverslip were washed three times with NaCl/P
i
, placed at

the opposite site o n a glass-slide w ith 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: Hybond
TM
-N, code RPN
303 N). The membrane was hybridized with the 612-bp
large dj2 fragment or with the entire Mydj2 cDNA labe led
with [a-
32
P] dCTP (NEN: 3000 CiÆmmol
)1
, NEG 513H) a s
indicated, washed, and exposed to Kodak XAR film at
)70 °C for 2–5 d ays using Kodak lightening plus screens.
RNA s tability a ssays were carried out as describe d [ 25].
Actinomycin D (Sigma: 10 mgÆmL
)1
in dimethylsulfoxide)
was added to COS and HeLa cells to a final concentration
of 10 lgÆmL
)1
. A fter 0, 1, 2, 3 a nd 4 h of incubation with
actinomycin D, cells (8 · 10
6
) were washed with NaCl/P

i
and total RNA w as prepared. Northern b lot analysis and
detection of both messages, large dj2 and small dj2 mRNAs,
were performed as described previously [34].
RESULTS
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 c DNA source we used the premade by Stratagene cDNA
library of monkey COS cells which was screened using the
entire hum an 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 A TG 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 m RNA having 0.9-kb
additional sequences at the 3¢ UTR ( Fig. 1A).
Further sequence comparison between the isolated mon-
key dj2 cDNA (clone 5aI) a nd 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, l eft panel), or the fragment
between 1517 and 2129 bp, in the 3¢ UTR region, which can
hybridize only to the large dj2 mRNA (Fig. 1 B, right panel).
Following our theoretical approach we examined the
expression of both d j2 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. T he 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 t his ratio
did not change when the cells were exposed to heat shock
(Fig. 1 B). When the 612 bp fragment of t he 3¢ UTR r egion
(1517–2129 bp) was used as a probe, only the large dj2
mRNA was i dentified (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, r evealed that both messages can
be detected in all tissues but their d istribution 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 t issues examined except skeletal
muscle. I n c ontrast large dj2 mRNA was f ound to be
abundant mainly in the brain, kidney and lung (Fig. 2A).
The major observation of this s tudy was t hat the large dj2
mRNA distribution differed from that of the small dj2 and
that the levels of both messages v aried according to t he c ell

or tissue t ype. The significanse of the above finding remains
to be clarified.
We then examined the distribution of t he corresponding
dj2 protein in the s ame rat and mice tissues (Fig. 2B,C,D).
For this study we used two d j2 specific antibo dies, one wh ich
Fig. 1. Characterization and c omparison of l arge and small dj2 mRNAs.
(A) Schematic re presentation and sequence comparison of t he m onkey
large dj2 cDNA and human small dj2 cDNA (B). Northern blot
analysis of the two forms o f d j2 m RNAs, using RNAs from c ontrol ( –)
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 po sition of 18S rRNAs is
shown at the bottom.
Ó FEBS 2002 Monkey dj2, a specific partner for hsc70 (Eur. J. Biochem. 269) 1555
recognizes the entire monkey dj2 protein (see Materials and
methods) a nd another that recognizes only the N-terminal
end (1–179 amino a cids) o f t he 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 l evels were
found to be particularly high in testis, brain, kidney and
liver. On th e other h and, 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 w ith 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, g iven 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 i ncubated periods, RNAs were p repared
and the samples were subjected to Northern blotting
analysis, using the full length M ydj2 cDNA as a probe.
Detection of the large a nd small Mydj2 mRNAs under the
conditions d escribed revealed that both messages were very
stable. Even 24 h after the treatment w ith actinomycin D,
both messages w ere present in detectable quantities
(Fig. 3A). It s hould 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 r eading frame o f the isolated
Mydj2 cDNA (clone 5aI) and detect the in vitro products
that our clone is able to produ ce, the dj2 (5aI) cDNA was
cloned in t he T7/T3 e xpression 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 i n 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)fromlung,brain,testis,kidney,heart,spleen,muscle,liverand
from COS and HeLa cells, were analyzed by Northern blotting, using
as prob e the
32
P-labeled c DNA 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 m ethods. Equal amo unts 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 rec ombinant Mydj2 prot ein. The lower
band observed in testis probably represents a testis spe cific 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
)1
actinomycin-D for 0, 2, 4, 16
and 24 h. (A) Parallel cultures were treated in the same way, with
actinomycin D and exp osed in heat shock for 90 m in at 43 °C.
(B) Then, RNAs were prepared and 2 0 lg of each sample were
subjected to RNA blot analysis using the entire Mydj2 cDNA, radio-
labelled with [c
32
P]dCTP, as a probe. Integrity o f RNAs was verified
by the a ppare ntly identical intensities of 1 8S rRNAs.

1556 P. Bozidis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
cells, 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 °Cfor
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 intrace llular 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 subc ellular 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 F ig. 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 t he
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 · 10
6
cells 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 c oprecipitated 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 ob tained when lysates from heat shock ed
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 d j2 form translocates to the cell nucleus. CV1
cells growing under physiological conditions (37 °C) or heat treated f o r

120 min at 4 3 °C followed by recovery at 37 °Cfor160min,were
collected and fractionated into whole cell, cytoplasmic and nuclear
extracts as described in Materials and methods. Control and heat
shocked extracts w ere then subjected to Western blotting analysis
utilizing an anti-dj2 Ig. P, denotes the purifi ed r ecombinant Mydj2
protein.
Fig. 4. Intracellular distribution of Mydj2 in CV1 c ells. Cells growing at
37 °C(B)ortreatedat43°C for 120 min followed by recovery a t
37 °C for 160 min (C), were fixed and processed for immunofluo rence
staining. (B) and (C), represent c ells stained with anti-dj 2 Ig. (A) rep-
resents cells stained with preimmune serum.
Ó FEBS 2002 Monkey dj2, a specific partner for hsc70 (Eur. J. Biochem. 269) 1557
Having 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 t hat, CV1 cells were har vested and RIPA cell extracts
from control and heat-shocked cells were prepared. T hese
extracts were immunoprecipitated with anti-dj2 or a nti-
hsp70 or anti-hsc70 specific Ig as described in Materials and
methods. The immunoprecipitated samp les were t hen
resolved by SDS/PAGE and s ubjected to Western blotting
using t he appropriate antibodies. As shown in Fig. 7, when
an anti-dj2 Ig was used for immunoprecipitation, only t he
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 u sed for immunopre-
cipitation (Fig. 7A). In contrast, immunoprecipitation with
an anti-hsp70 specific Ig revealed that dj2 does not associate
with the hsp70 protein (Fig. 7B).

The a bove results clearly demonstrate t hat 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.
DISCUSSION
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 c ombination and regulation of all these nanoma-
chines. However, we know that chaperones play an essential
role in various cellular functions, such a s the acquisition o f
thermotolerance and ce ll 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 4 0-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 g ives 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 p robe and RNAs
from cell lines, w e identified two different in size mRNAs.
The s teady-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 l evels of both m essages varied in all cells
and tissues examined.
Our study showed that the long 3 ¢ UTR d id not
contribute to the message stability under normal or heat
shocked conditions, given that no known sequences that
were responsible of regulating the m essage stability were
found. This was in a greement 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 a nd 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 °Cfor
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 immuno precipitates were then su bjec ted 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, im munoprecipitate of control lysate; lane 5, immuno-
precipitate of heat s hocked 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 h eat-shocked cells were submitted to a pull-
down assay using His-dj2 on Ni/nitrilotriacetic acid agarose b eads as

the binding substrate. Cell extracts, washes and eluted protein samples
were analyzed by Western blotting using specific a ntibodies against
hsp70, hsc70 and Mydj2. 1,4,1¢,4¢:cellextracts,2,5,2¢,5¢: third wash-
ings, 3,6,3¢,6¢: eluted p roteins.
1558 P. Bozidis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
According to our findings only the nonfarnesylated form of
dj2 is a ble to t ranslocate into the nucleus. In contrast, the
farnesylated form remains localized to the cytosolic fraction.
Moreover during heat shock (90 min at 4 3 °Cand60min
recovery to 37 °C), the farnesylated dj2 protein translocates
mostly to the nucleus suggesting that t his migration is related
to the facilitation of t he folding of the he at denatu red nuclear
proteins. This r esult is i n agreement with previous observa-
tion which s uggests t hat t he 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 o f
denatured l uciferase [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-d own assays using
recombinant Mydj2 fused t o 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 d j2, 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, w e
tried to repeat the same experiments under semi in vivo
conditions. Indeed, we once again identified the existence o f
hsc70/dj2 c omplexes in cell e xtracts b y 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 a nd
dj2 constitute potent partners in the construction of a
functional chaperone pair.
ACKNOWLEDGEMENTS
We thank Dr S. Kato for the gift o f t he human dj2 cDNA clone, Dr
Vezyraki for her special contribu tion on anim al 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 Institutio n (11-7-2000) an d partially f rom an EU grant
(QLRT-1999, #30720).
REFERENCES
1. Hartl, F U. (1996) Molecular chaperones in cellular protein
folding. Nature 381, 571–579.
2. Schmid, D., Baici, A., Gehring, H. & Christen, P. (1994) Kinetics
of molecular chaperone action. Science 263, 971–973.
3. Laufen,T.,Mayer,M.,Beisel,C.,Klostermeier,D.,Mogk,A.,
Reinstein, J. & Bukau, B . (1999) Mechanism of regulation o f
hsp70 chaperones by DnaJ co-chaperones. Proc. Natl Acad. Sci.
USA 96, 5452–5457.
4. Ohtsuka, K. & Hata, M. (2000) M ammalian Hsp40/DnaJ
homologs: cloning of novel cDNAs and a proposal for their
classification and nomenc lature. Cell Stress Chaperones 5, 98–112.
5. Ohtsuka, K. (1993) Cloning of a cDNA for heat-shock protein

hsp40, a human homologue of bacterial DnaJ. Biochem. Biophys.
Res. Commun. 19 7, 235–240.
6. Raabe, T. & Manley, J.L. (1991) A human homologue of the
Escherichia coli DnaJ heat shock protein. Nucleic Acids Res. 19,
6645.
7. Chellaiah. A., Davis, A. & Mohanakumar, T. (1993) Cloning of a
unique human homologue of the Escherichia coli DNAJ heat
shock protein. Biochim. Biophys. Acta 1174, 11–113.
8. Oh, S., Iwahori, A. & K ato, S. (1993) Human c DNA encoding
DnaJ protein homologue. Biochem. Biophys. Acta. 1174, 114–116.
9. Cheetham, M.E., Brian, J.P. & A nderton, B.H. ( 1992) Human
homologues of the bacterial heat-shock protein DnaJ a re pre-
ferentially expressed in neurons. Biochem. J. 284, 469–476.
10. Terada, K. & M ori, M. (2000) Characterization of human DnaJ
homologs dj2 and dj3, and bag-1 are positive cochaperones of
hsc70. J. Biol. Chem. 275 , 24728–24734.
11. Kanazawa, M., Terada, K., Kato, S. & Mori, M. (1997) HSDJ, a
human homolog of DnaJ, is f arn esylated and is involved in protein
import into mitochondria. J. Biochem. 121, 890–895.
12. Szabo, A.R., Korszun, F.U., Hartl & Flanagan, J . (1996) A zinc
finger-like domain of the molecular chaperone DnaJ is involved in
binding to denatured protein s ubstrates. EMBO J. 15 , 408–417.
13. Karzai, A.W. & McMacken, R. (1996) A bipartite signaling
mechanism involved in D naJ-mediated activation o f the Escheri-
chia coli DnaK protein. J. Biol. Chem. 271, 11236–11246.
14. Cheetham, M.E. & Caplan, A.J. (1998) S tructure, function and
evolution of DnaJ: conservation a nd adaptation of chaperone
function. Cell Stress Chaperones 3, 28–36.
15. Caplan, A.J., Tsai, J., Casey, P.J. & Douglas, M.G. (1992) Far-
nesylation of YDJ1p is required for function at elevated growth

temperatures in Saccharomyces cerevisiae. J. Biol. Chem. 267,
18890–18895.
16. Glomset, J.A., Gelb, M.H. & Farnsworth, C.C. (1990) Prenyl
proteins in eukaryotic cells: a new type of membrane anchor.
Trends Bioc hem. Sci. 15, 139–142.
17. Tang, Y., Ram akrish nan, C., Thomas, J. & DeFranco, D.B .
(1997) A role for HDJ-2/HSDJ in correcting subnuclear traffick-
ing, transactiva tion, and t ransrepression defects of a glucocorti-
coid receptor zing finger mutant. Mol. Biol. Cell. 8, 795–809.
18. Caplan, A.J., Cyr, D.M. & Douglas, M.G . (1992) YDJ 1p facil-
itates polypeptide translocation across different intracellular
membranes by a conserved mechanism. Cell 71 , 1143–1155.
19. Terada, K., Kanazawa, M., Bukau, B . & Mori. M. (1997) The
human DnaJ homologue dj2 facilitates mitochondrial protein
import and lucifera se refolding. J. Cell Biol. 139, 1089–1095.
20. Meacham,G.C.,Lu,Z.,King,S.,Sorscher,E.,Tousson,A.&
Douglas, M.C. (1999) The Hdj-2/Hsc70 chaperone pair facilitates
early steps in CFTR biogenesis. EMBO J. 18, 1492–1505.
21. Wytte nbach, A., C armichael, J ., Swartz, J ., Furlong, R.A.,
Narain, Y., Rankin, J. & Rubinsztein, D.C. (2000) Effects of heat
shock, heat shock protein 40 (HDJ-2) a nd proteosome inhibition
on protein aggregation in cellular models of Huntington’s disease.
Proc. N atl Acad. Sci. USA 97, 2998–2903.
22. Kobayashi,Y.,Kume,A.,Li,M.,Doyu,M.,Hata,M.,Ohtsuka,
K. & Sobue, G. (2000) Chaperones hsp70 and hsp40 s upress
aggregate formation and apoptosis in cultured neuronal cells
expressing truncated androgen receptor protein with expanded
polyglutamine tract. J. Biol. Chem. 275, 8772–8778.
23. Royaux, I., M inne r, F., G offinet, A.M. & Lampert de Ro uvroit, C.
(1998) A DnaJ-like gene, Hsj2, maps to mouse chromosome 5, at

approximately 24 cM f rom the centromere,. Genomics 53,415.
24.Ohtsuka,K.&Hata,M.(2000)MammalianHSP40/DNAJ
homologs: cloning a novel cDN A and a proposal for the ir clas-
sification and nomenclature. Cell Stress Chaperones 5, 98–112.
25. Kritzik, M.R., Ziober, A.F., Dicharry, S., Conrad, D.J. & Sigal, E.
(1997) Characterization and sequence of an additional 15-lipoxy-
genase transcript and of the human gene. Biochim. Biophys. Acta
1352, 267–281.
26. Angelidis, C .E., Nova, C., Lazaridis, I., Kontoyiannis, D., Kollias,
G. & Pagoulatos, G.N. (1996) Overexpression of HSP70 in
transgenic mice results in increased cell thermotolerance. Trans-
genics 2, 111– 117.
Ó FEBS 2002 Monkey dj2, a specific partner for hsc70 (Eur. J. Biochem. 269) 1559
27. Angelidis, C .E., Lazaridis, I. & Pagoulatos, G.N. (1988) Specific
inhibition of simian virus 40 protein synthesis by heat and arsenite
treatment. Eur. J. Biochem. 179, 27–34.
28. Tanguay, R.M., W u, Y. & K handjian, E.W. (1993) Tissue-specific
expression o f heat shock proteins of the mouse in t he absence o f
stress. Dev. Genet. 14, 112–118.
29. Angelidis, C.E., Lazaridis, I. & Pagoulatos, G .N. (1999)
Aggregation of h sp70 and hsc70 in vivo is distinct and tempera-
ture-dependent and their chaperone function is directly related to
non-aggregated forms. Eur. J. Biochem. 259, 505–512.
30. Davis, A.R., Alevy, Y .G., Chellaiah, A., Quinn, M.T. &
Mohanakumar, T. (1998) Characterization of HDJ-2, a human 40
kD heat shock p rotein. Int. J. Bioc hem. Cell Biol. 30, 1203–1221.
31. Hohfeld, J., Minami, Y. & Hartl, F U. (1995) Hip, a novel
cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction
cycle. Cell 83 , 589–598.
32. Zeiner, M., Gebauer, M. & Gehring, U. (1997) Mammalian

protein RAP46: an interaction partner and m odulator of 70 kDa
heat shock p roteins. EMBO J. 16, 5483–5490.
33. Chomozynski, P. & Sacchi, N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyan ate-pH clo roform extrac-
tion. Anal. B iochem. 162, 156–159.
34. Sainis, I., Angelidis, C.E., Pagoulatos, G.N. & Lazaridis, I. (1994)
The hsc70 gene, which is sli ghtly induced b y heat, is the main virus
inducible member of the hsp70 gene f amily. FEBS Lett. 35 5,
282–286.
35. Lazarov, M.E., Martin, M.M., Willardson, B.M. & Elton, T.S.
(1999) Human phosducin-like protein (hPhLP) m essenger RNA
stability is regulated by cis -acting instability elements present in t he
3¢-untraslated region. Biochim. Biophys. Acta 1446, 253–264.
36. Li, G.C. & Werb, Z. (1982) Correlation between synthesis of heat
shock p roteins and development of thermotolerance in Chinese
hamster fibroblasts. Proc. Natl Acad. Sci. USA 79, 3219–3222.
37. Angelidis, C.E., Lazaridis, I. & Pagoulatos, G.N. (1991) Con-
stitutive expression of heat-shock p rotein 70 i n mammalian c ells
confers thermoresistance. Eur. J. Biochem. 199, 35–39.
38. Plumier, J.C.L., Ross, B.M., Currie, R.W., Angelidis, C.E.,
Kazlaris, H., Kollias, G. & Pagoulatos, G.N. (1995) Trans-
genic mice expressing the human heat shock p rotein 70 have
improved post ischemic m yocardial recovery,. J. Clin. Invest. 95,
1854–1860.
39. Morimoto, R.I. (1991) Heat shock: the role of transient inducible
responses i n cell damage. Trans. Diff. Cancer Cells 3, 296–301.
40. Leppa, S. & Sistonen, L. (1997) Heat shock response-patho-
physiological impl ications. The Finnish Med. Soc. DUODECIM,
Ann. Med. 29, 73–78.
41. Rokutan, K., Hirakawa, T., Teshima, S., Nakano, Y., Miyoshi,

M., Kawai, T., Konda, E., Morinaga, H., Nikawa, T. & Kishi, K.
(1998) Implications of heat shock/stress proteins for medicine and
disease. J. Med. Invest. 44, 137–147.
42. Latchman, D. ( 1998) Heat shock proteins: Protective effect and
potential therapeu tic use. Int. J. Mol. Med. 2, 375–381.
43. Chai, Y., Koppenhafer, S.L., Bonini, N.M. & Paulson, H.L.
(1999) Analysis of the role of heat shock protein (Hsp) molecular
chaperones in polyglutamine disease. J. Neurosci. 19, 10338–
10347.
1560 P. Bozidis et al. (Eur. J. Biochem. 269) Ó FEBS 2002