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: chaggeli@cc.uoi.gr 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. 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(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 . Mydj2 as a potent partner of hsc70 in mammalian cells Petros Bozidis, Ioannis Lazaridis, Gerassimos N. Pagoulatos and Charalampos E. Angelidis Laboratory. bacterially express Mydj2 the corresponding cDNAwasamplifiedbyPCRusingprimerI(5¢-GCA GTAGAGGATCCTGAAAGAAA-3¢) and primer II (5¢-GTTATTCAGTCGACCATTAAGAGG-3¢)