Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration Hideaki Itoh 1 , Atsushi Komatsuda 2 , Hiroshi Ohtani 2 , Hideki Wakui 2 , Hirokazu Imai 2 , Ken-ichi Sawada 2 , Michiro Otaka 3 , Masahito Ogura 1 , Akira Suzuki 1 and Fumio Hamada 4 1 Department of Biochemistry, 2 Department of Third Internal Medicine, and 3 Department of First Internal Medicine, Akita University School of Medicine, Akita City, Japan; 4 Department of Material-Process Engineering and Applied Chemistry for Environment, Akita University Faculty of Engineering and Resource Science, Akita City, Japan There are few reports concerning the sorting mechanisms of mammalian HSP60 into the mitochondria from the cyto- plasm. In the present study we investigated the protein import system. Based on immunoblotting and immuno- histochemistry, HSP60 was detected in both the cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay. HSP60 mRNA was increased in the kidney papilla of rats that had been water restricted for three and five days, but no changes in HSP60 mRNA were detected in the cortex or the medulla of the rat kidneys. Upon immunoblotting, HSP60 was detected in both the cytoplasm and the mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the kidney papilla of rats that were water restricted but the protein was increased in the mitochondria of the rat kidney papilla. We also analysed binding of the protein to the signal sequence of HSP60 using signal sequence-affinity column chromatography. We identified only one protein band with a molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from the affinity column by an excess of signal peptide or by 5 m M ATP. Upon immunoblotting, the 70-kDa pro- tein cross-reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytoplasmic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP70. Keywords: HSP60; HSP70; molecular chaperone; protein sorting. In both prokaryotic and eukaryotic cells the misfolding and aggregation of proteins during biogenesis, and under conditions of cellular stress, are prevented by molecular chaperones (reviewed in [1–3]). It is now generally accepted that molecular chaperones are required for the correct folding assembly both of misfolded proteins and of newly synthesized polypeptides. The chaperonin GroEL/GroES is the only chaperone system in Escherichia coli that is essential for the growth [4]. GroEL is an oligomeric double-ring complex consisting of 14 identical 58-kDa subunits that form a cylindrical structure with two large cavities. Cochaperone GroES contains seven identical 10-kDa subunits assembled as one heptameric ring and binds to the apical GroEL domains [5]. The chaperonin mediates the folding of the polypeptide chain in an ATP-dependent reaction [6]. In contrast with GroEL, very little is known about the structure and physiological functions of the mammalian chaperonin homologue HSP60. Mammalian HSP60 was first reported as a mitochondrial P1 protein [7]. Gupta and coworkers were the first to clone and sequence the protein, and the deduced amino acid sequence showed a strong homology to GroEL and the 65-kDa major antigens of mycobacteria. For these reasons, it was believed that HSP60 may have functions only in the mitochondria and that there is no chaperonin homologue in the cytoplasm of eukaryotic cells. It has been shown that the chaperonin- containing t-complex polypeptide 1 (CCT), also called TriC, assists in the folding of actin and tubulin in the presence of ATP in vitro and binds newly synthesized actin and tubulin in vivo [8,9]. CCT/TriC has a double-torus-like structure with an eight-fold rotational symmetry assem- bled from 16 subunits [10]. In mammalian somatic cells, CCT/TriC is composed of eight different subunits of  60- kDa each [11]. Although CCT/TriC is a member of the chaperonin family that includes GroEL and HSP60, the sequence homology between CCT/TriC and GroEL is < 40% [11]. For these reasons, it has been generally believed that the mammalian cytoplasmic and mitochond- rial chaperonin are CCT/TriC and mitochondrial HSP60 (P1 protein), respectively. We have purified a functional HSP60 from rat liver cytoplasm and mitochondria [12]. In amino acid sequence analysis, cytoplasmic HSP60 had an N-terminal signal sequence which is not present on mitochondrial HSP60. Both proteins showed chaperone activity in vitro. We have reported that cytoplasmic HSP60 may function as an immunophilin [13]. The major targeting protein of an Correspondence to H. Itoh, Department of Biochemistry, Akita University School of Medicine, 1 1 1 Hondo, Akita City, 010 8543, Japan. Fax: + 81 18 884 6078, Tel.: + 81 18 884 6078, E-mail: hideaki@med.akita-u.ac.jp Abbreviations: PDI, protein disulfide isomerase; CS, citrate synthase; G3PDH, glyceraldehydes-3-phosphate dehydrogenase. (Received 26 August 2002, revised 7 October 2002, accepted 15 October 2002) Eur. J. Biochem. 269, 5931–5938 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03317.x immunosuppressant mizoribine is cytoplasmic HSP60. These results suggest that HSP60 is not quickly imported into the mitochondria after being synthesized in the cytoplasm. Recently, it has been shown that cytoplasmic HSP60 forms a macromolecular complex with Bax and Bak in vitro [14].HSP60mayplayakeyrolein antiapoptosis in the cytoplasm. It has also been reported that HSP60 exists in human plasma, and there was evidence of an association between the levels of HSP60 in the plasma and the proinflammatory cytokine, tumour necrosis factor a, and with various psychosocial measures [15]. In the mammalian cytoplasm, HSP60 may play important roles including chaperone activity, immunophi- lin, and antiapoptosis. HSP60 will be rapidly imported into the mitochondria when these functions are required in the mitochondria. In the present study, we investigated the mammalian HSP60 import system into the mitochondria. Almost all of the HSP60 was imported into the mitochondria in the kidney papilla of water-restricted rats; there were no changes in protein distribution in the cortex and papilla. Cytoplasmic HSP70 was detected as a protein binding specifically to the signal sequence of HSP60. Sorting mechanisms of the mammalian HSP60 are discussed. MATERIALS AND METHODS Materials Rat liver cytoplasm, mitochondria, microsome, and nucleus were subcellularly fractionated as described previously [12]. Activated CH-Sepharose 4B was from Amersham Phar- macia Biotech. The rat glyceraldehyde-3-phosphate dehy- drogenase (G3PDH) RT/PCR control kit was from Clontech. 5-Bromo-4-chloro-3-indolyphosphate p-toluidine salt and nitroblue tetrazolium chloride were from Roche Diagnostics. Antibodies against HSP70, HSP90 and HSP60, respectively were used as described previously [12,16,17]. Antibodies against cytochrome c and citrate synthase were purchased from Sigma and Chemicon International, Inc., respectively. Antibodies against PDI (protein disulfide isomerase) and Histon H3 were pur- chased from Santa Cruz Biotechnology, Inc. The signal sequence of humanHSP60 (MLRLPTVFRQ MRPVSRVLAP HLTRAY) was synthesized by solid phase techniques, and an antibody against the signal sequence of human HSP60 was produced using a synthetic peptide as described [12]. The protocols for animal experimentation described in this paper were previously approved by the Animal Research Committee, Akita University School of Medicine; the ÔGuidance for Animal ExperimentationÕ of the University was completely adhered to in all subsequent animal experiments. Cytoplasmic and mitochondrial HSP60 were purified from porcine liver as described [12]. The rat Cpn10 (HSP10) expression vector (pRSC550-Cpn10) was kindly provided by D. J. Naylor (The University of Adelaide, Australia). Recombinant rat HSP10 was expressed and purified as described [18]. Mitochondrial import of HSP60 in vitro The purified cytoplasmic and mitochondrial HSP60 were labelled with 125 I using an IODO-GEN iodination reagent (PIERCE). The labelled HSP60 was incubated with or without isolated rat liver mitochondria (0.5 mgÆmL )1 )and/ or 5 m M MgCl 2 /ATP in 10 m M Tris/HCl pH 7.4 for 60 min at 37 °C. After incubation, the samples were centrifuged for 10 min at 15 000 g. The supernatant was used as the supernatant for SDS/PAGE. The precipitates were washed with 10 m M Tris/HCl pH 7.4 and centrifuged for 5 min at 15 000 g. The precipitates were dissolved in SDS sample buffer and used for SDS/PAGE. The supernatant and precipitates were analysed on SDS/PAGE (6.5% polyacryl- amide gel), followed by autoradiography. Measurement of protein aggregation The influence of HSP60 in the presence or absence of HSP10 and ATP during the thermal aggregation of mitochondrial citrate synthase (CS; Boehringer-Mannheim) at 43 °C was monitored as described [19]. To monitor the thermal unfolding/aggregation, the CS concentration was 0.075 l M in 40 m M Hepes buffer pH 7.4 in the presence or absence of purified porcine HSP60 (0.075 l M ), recombinant rat HSP10 (0.075 l M ), and ATP/MgCl 2 (5 m M ). The light scattering of CS was monitored over 60 min by the optical density at 500 nm using a Pharmacia Ultrospec 3000 UV– Vis spectrophotometer equipped with a temperature control unit with semimicro-cuvettes (1 mL) having a path length of 10 mm. In this study, 1 arbitrary unit denotes an absorb- ance of  0.2 at 500 nm. RNA preparation and RT/PCR Total rat kidney RNA was reverse transcribed in a reaction volume of 20 lL using 500 ng oligo (T) 15 and 200 U SuperScript II reverse transcriptase (Gibco BRL), 0.5 m M each of the four dNTPs in 50 m M Tris, pH 8.3, 75 m M KCl, 3m M MgCl 2 and 10 m M dithiothreitol for 1 h at 42 °C. The cDNA was amplified using the rat HSP60 sense primer (5¢-CAAATGAAGAGGCTGGGGATGGCA-3¢) and antisense primer (5¢-GAGCAGGTACAATGGACT GAACAC-3¢) in a 50-lL reaction volume containing 200 l M each of the four dNTPs and 2.5 U Taq polymerase (Gibco BRL) to obtain partially coded cDNA (467 bp) as described previously [20]. The rat G3PDH RT-PCR control kit (Clontech) was used as a control in the experiment. Water-restricted rat Male Wister rats weighing about 150 g were purchased from the Sizuoka Agriculture Cooperative Association for Laboratory Animals, Hamamatsu, Japan. Nine rats were fed a commercial rat chow replete with add dietary requirement and were given free access to water and food for 7 days before water-restriction. They were then divided into three groups. Three rats in group 1 were used as the control. Three rats in groups 2 and 3 were restricted to water in a tube for 3 and 5 days, respectively. Urine was collected from each rat (in groups 1, 2 and 3 on days 0, 3 and 5, respectively) and the urinary volume and osmolarity were measured. Immediately after urine collection, blood was taken from the subclavian vein of each rat for the measurement of serum creatinine and blood urea nitrogen. Therefore, water-restricted rat kidneys were then obtained from these rats. 5932 H. Itoh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Subcellular fractionations of rat livers or water-restricted rat kidneys All operations were carried out at 0–4 °C. The livers were homogenized with buffer (10 m M Tris/HCl, pH 7.4, 0.25 M sucrose, 0.1 m M EDTA). After centrifugation at 600 g for 5 min, the precipitate was discarded. The 600 g supernatant was further centrifuged at 7000 g for 10 min, and the supernatant (S1) and precipitate (P1) were treated by further centrifugation. The precipitate (P1) was dissolved in the buffer and centrifuged at 5000 g for 10 min. The 5000 g precipitate was used as the mitochondrial fraction. The supernatant (S1) was centrifuged at 54 000 g for 60 min, and the supernatant was further centrifuged at 105 000 g for 60 min The 105 000 g supernatant was used as the cytoplasm. The water-restricted rat kidneys were divided into three segments (cortex, medulla and papilla) and homogenized with buffer (10 m M Tris/HCl, pH 7.4, 0.25 M sucrose, 0.1 m M EDTA). The homogenates were subcellularly fractionated as described above. Each segment of the water-restricted rat kidneys was used for RT-PCR or immunoblotting. Affinity column chromatography A signal sequence affinity column was prepared using the synthetic peptide and activated CH-Sepharose 4B (Amer- sham Pharmacia Biotech) according to the instruction manual. Rat liver was homogenized with 10 m M Tris/ HCl, pH 7.4, 0.25 M sucrose and 0.1 m M EDTA. The 105 000 g supernatant was used as the cytoplasm as described above. The rat liver cytoplasm was applied onto the signal sequence affinity column pre-equilibrated in 10 m M Tris/HCl pH 7.4 and washed with 10 column vols of the buffer containing 0.5 M NaCl. After washing the column, binding proteins were eluted from the column with the 0.1, 1 and 5 m M signal peptide of HSP60 or 5 m M ATP in the same buffer. The eluants were analysed by SDS/PAGE [21] or by immunoblotting [22]. Gel electrophoresis and immunoblotting SDS/PAGE was carried out according to the procedure of Laemmli using 6.5–10% polyacrylamide gels. After electro- phoresis, gels were stained with 0.1% Coomassie Brilliant Blue R250 in a mixture of 25% (v/v) isopropyl alcohol and 10% (v/v) acetic acid and destained with 10% (v/v) isopropyl alcohol and 10% (v/v) acetic acid. Proteins were then transferred electrophoretically to a polyvinylidene difluoride membrane and processed as described by Towbin et al. [22]. After incubation with antibodies against HSP60, the signal sequence of HSP60, HSP90, cytochrome c,and citrate synthase (diluted 1 : 500 to 1 : 1000 in 7% (w/v) skim milk), each membrane was treated with alkaline phosphatase–conjugated anti-rabbit IgG (Bio-Rad) (diluted 1 : 1000 in 7% (w/v) skim milk) or anti-mouse IgG (bioRad) (diluted 1 : 1000 in 7% (w/v) skim milk). The antigen–antibody complexes were visualized by reacting the bound alkaline phosphatase with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyphosphate p-tolui- dine salt. Electron microscopic immunohistochemistry Ultrathin sections of rat kidneys were obtained as described previously [12]. The sections were stained by the immuno- gold/silver staining method for electron microscopy using a silver enhancing kit (BioCell Research Laboratories). The sections were incubated with antibody against either the signal sequence of HSP60 or HSP60. The sections were then incubated with gold-labelled anti-rabbit IgG (Nanoprobes, New York, USA) for 1 h, and the sections were finally incubated with the silver developer of the enhancing kit. RESULTS Localization of HSP60 in mammalian organs Mammalian HSP60 has a signal sequence of 26 amino acid residues at the N terminus. In the present study, we used two different types of antibodies against HSP60; an antibody against the signal sequence of HSP60 and an antibody against cytoplasmic HSP60. At first, we examined the specificity of these antibodies using purified HSP60. There were slight differences in the migration of cytoplasmic and mitochondrial HSP60 (Fig. 1A): mitochondrial HSP60 migrated faster than cytoplasmic HSP60. The difference in migration is due to the signal sequence (M r ¼ 2926.8). As shown in Fig. 1B, an antibody against cytoplasmic HSP60 reacted with both cytoplasmic and mitochondrial HSP60. An antibody against the signal sequence of HSP60 reacted with cytoplasmic HSP60 only. We studied the localization of HSP60 in the unstressed rat kidney. Electron microscopic immunohistochemistry was performed. As shown in Fig. 1D, an antibody against the signal sequence of HSP60 mainly detected HSP60 in the cytoplasm. An antibody the against cytoplasmic HSP60 antibody detected HSP60 in both the cytoplasm and mitochondria (Fig. 1E). We next investigated the localization of HSP60 in vivo. Rat livers were subcellularly fractionated into four fractions (cytoplasm, microsome, mitochondria, and nucleus). The mitochondrial marker proteins, cytochrome c and citrate synthase, were detected only in the mitochondrial fractions (Fig. 1I and J). No protein bands were detected in the cytoplasm, microsome, or nucleus. On the contrary, a cytoplasmic marker protein, HSP90, was observed only in the cytoplasmic fraction (Fig. 1K). The microsomal and nuclear marker proteins, PDI and Histon H3, were detected only in the microsomal and nuclear fractions (Fig. 1L and M). These results suggested that the purity of each subcellular fraction was high. An anticytoplasmic HSP60 antibody reacted with HSP60 not only in the mitochondria but also in the cytoplasm (Fig. 1G). The quantities of cytoplasmic and mitochondrial HSP60 were almost equal. On the contrary, an antibody against the signal sequence of HSP60 was cross-reacted with cytoplasmic HSP60, but not with mitochondrial HSP60 (Fig. 1H). Thus, an antibody against the signal sequence of HSP60 is able to recognize only the cytoplasmic HSP60. Based on the results shown in Fig. 1 mammalian HSP60 exists in the mitochondria as mitochondrial HSP60. The protein also exists in the cytoplasm as a cytoplasmic HSP60 which has an N-terminal signal sequence. These results suggested that cytoplasmic HSP60 is stable in the cytoplasm Ó FEBS 2002 HSP60 sorting system (Eur. J. Biochem. 269) 5933 and that its sorting time into the mitochondria is quite different from those of other mitochondrial proteins such as cytochrome c and citrate synthase. In vitro HSP60 import We investigated the HSP60 import system of the mitochondria in vitro. As described in Materials and methods, isotope-labelled recombinant HSP60 was incu- bated with rat liver mitochondria in the presence or absence of ATP. In the absence of ATP, cytoplasmic HSP60 and mitochondrial HSP60 were both detected in the supernatant of the mitochondria (Fig. 2). Although mitochondrial HSP60 was detected in the supernatant of the mitochondria, cytoplasmic HSP60 was detected only in the precipitate of the mitochondria in the presence of ATP. However, the protein could not be imported into the mitochondria at 4 °Corat37°C in the absence of ATP (data not shown). These results suggest that the cytoplasmic HSP60 (having a signal sequence) would be imported into the mitochondria under appropriate con- ditions in vitro. Influence of HSP60 on protein aggregation To analyse the functional activity of cytoplasmic HSP60, we studied its action in protein folding and unfolding reactions Fig. 1. Specificity of antibodies and subcellular localization of HSP60 in rat livers. Purified cytoplasmic HSP60 and mitochondrial HSP60 were separated by on SDS/PAGE (6.5% polyacrylamide gel) followed by Coomassie Brilliant Blue staining (A), by immunoblotting with an anti-(cytoplasmic HSP60) Ig (B), or immunoblotting with an anti-(signal sequence HSP60) Ig (C). Lane 1, Purified mitochondrial HSP60; lane 2, purified cytoplasmic HSP60; lane 3, molecular standard proteins. Normal rat kidney sections were stained by the immuno-silver staining method using an anti- serum against the signal sequence of HSP60 (D) or an antiserum against cytoplasmic HSP60 (E). Arrows in all panels indicate the localization of HSP60. C, Cytoplasm; M, mitochondria. Rat livers were subcellularly fractionated into four fractions (cytoplasm, microsome, mitochondria and nucleus), and each fraction was electrophoresed on 9% or 6.5% SDS/polyacrylamide gels, which were stained with Coomassie Brilliant Blue (F), or immunoblotted with: anti-(cytoplasmic HSP60) Ig (G), an anti-(signal sequence HSP60) Ig (H), an anti-(cytochrome c)Ig(I), ananti-CSIg(J),anti-HSP90Ig(K),anti-PDI Ig (L), or anti-(Histon H3) Ig (M). Lane 1, Cytoplasm; lane 2, microsome; lane 3, mitochondria; lane 4, nucleus; and lane 5, molecular standard proteins. Fig. 2. Mitochondrial import of HSP60 in vitro. The purified cyto- plasmic and mitochondrial HSP60 were labelled with 125 Iandincu- bated in the presence or absence of mitochondria and ATP/Mg as described in Materials and methods. After centrifugation, the super- natant and precipitate were analysed by SDS/PAGE (6.5% poly- acrylamide gel) followed by autoradiography. c60, Cytoplasmic HSP60; m60, mitochondrial HSP60; S, supernatant; P, precipitate. 5934 H. Itoh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 in vitro. As an assay system, the thermal unfolding and aggregation of the mitochondrial CS was used, because CS is inactivated and rapidly aggregates upon incubation at 43 °C [20,23]. As shown in Fig. 3, spontaneous aggregation occurred at 43 °C. The purified cytoplasmic HSP60 and recombinant HSP10 in the presence of ATP almost completely inhibited thermal aggregation of CS. Only HSP60 or HSP60/HSP10 in the absence of ATP showed less effect on the thermal aggregation of CS. As a consequence, CS is effectively stabilized in the presence of HSP60/HSP10/ATP. In vivo HSP60 sorting into mitochondria As mentioned above, mammalian HSP60 is not always quickly imported into the mitochondria after being syn- thesized on free ribosomes in the cytoplasm of unstressed organs. We investigated the sorting conditions of mamma- lian HSP60 in vivo. In the present study, we used kidneys from water-restricted rats (Fig. 4). Rats were water-restric- ted for 3 or 5 days and then the kidneys were separated into cortex, medulla, and papilla. Compared with the kidneys of normal rats, the osmotic pressure in the kidneys of water- restricted rats was increased about 10 times (data not shown). Although G3PDH mRNA was stable in all kidney sections, HSP60 mRNA was increased in the papilla of the rat kidneys after 3 and 5 days of water restriction. No changes in the HSP60 mRNA were detected in the cortex and medulla in the rat kidneys after 3 and 5 days of water restriction. We investigated the quantity of HSP60 in the cyto- plasm and mitochondria by immunoblotting. No changes in the quantity and localization of HSP90 were observed under the severe conditions (Fig. 4E). The same data were obtained from the mitochondrial marker proteins cytochrome c (Fig. 4F) and CS (Fig. 4G). HSP60 was detected both in the cytoplasm and mitochondria of the water-restricted renal cortex and medulla. However, no changes in the quantity and localization of the protein were observed (Fig. 4C). On the contrary, HSP60 was remarkably decreased in the cytoplasm and increased in the mitochondria in the water-restricted renal papilla (Fig. 4C). The results were identical to the changes in the HSP60 mRNA in the cortex, medulla, and papilla. Taken together, HSP60 is synthesized and stably localized in the cytoplasm under unstressed conditions, and HSP60, induced in the cytoplasm under severe stress conditions such as water restriction, is quickly imported into the mitochondria in vivo. Investigation of proteins binding to the signal sequence of HSP60 We investigated the proteins binding to the signal sequence of HSP60 using signal sequence affinity column Fig. 3. Measurement of protein aggregation. Thermal aggregation of CS (0.075 l M ) in the absence of additional components (s), in the presence of an equal molar ratio of HSP60 (n), an equal molar ratio of HSP60/HSP10 (e), an equal molar ratio of HSP60 and 5 m M ATP/ Mg (m), and an equal molar ratio of HSP60/HSP10 and 5 m M ATP/ Mg (r) was monitored at 500 nm as described in Materials and methods. Fig. 4. In vivo import system of HSP60. Three or 5 day water- restricted rat kidneys were separated into cortex, medulla and papilla. The total RNA was reverse-transcribed, and the cDNA was amplified using rat HSP60 sense and antisense primers or a rat G3PDH control kit. (A) HSP60 mRNA. (B) G3PDH mRNA. The separated renal cortex, medulla, and papilla were subcellularly fractionated into cytoplasm and mitochondria. Samples were developed on SDS/ PAGE, followed by immunoblotting with: an anti-(cytoplasmic HSP60) Ig (C), an anti-(signal sequence HSP60) Ig (D), an anti-HSP90 Ig (E), an anti-(cytochrome c) Ig (F), or an anti-(citrate synthase) Ig (G). In panels C, D, E, F and G, C and M denote cytoplasm and mitochondria, respectively. In all panels, 0, 3 and 5 denote water restriction for 0, 3 and 5 days. Ó FEBS 2002 HSP60 sorting system (Eur. J. Biochem. 269) 5935 chromatography. After washing the column, the proteins were eluted with an excess of the signal peptide. Only one protein band, with a molecular mass of 70 kDa, was detected on SDS/PAGE (Fig. 5A). The 70-kDa protein was also eluted from the affinity column by a linear gradient of the signal peptide (Fig. 5B). No other proteins bands were observed in the eluant. We also analysed the binding proteins by other elution methods. Proteins were eluted from the column with 5 m M ATPandthesame70- kDa protein band was detected in the eluant. To identify the 70-kDa protein, we analysed its reactivity with an anti-HSP70 Ig by using an immunoblotting analysis. The protein eluted by the signal peptide or ATP reacted with the anti-HSP70 Ig (Fig. 5D) suggesting that the protein binding to the signal sequence of HSP60 is cytoplasmic HSP70. DISCUSSION Mammalian HSP60 cDNA was first cloned as a mito- chondrial P1 protein [7]. For these reasons, it has long been believed that mammalian HSP60 is located and functions only in the mitochondria. We previously reported the purification and characterization of HSP60 from the rat liver cytoplasm and mitochondria [12]. Cytoplasmic HSP60 has a 26-amino acid signal sequence at the N terminus of the protein which is highly degenerate and is capable of folding into a positively charged amphiphilic helix. On the con- trary, mitochondrial HSP60 does not have this sequence. Although the antibody against cytoplasmic HSP60 was recognized by both the cytoplasmic and the mitochondrial HSP60, an antibody against the signal sequence of HSP60 was recognized only by the cytoplasmic HSP60 in the immunoblotting analysis. However, the antibody cross- reacted mainly with HSP60 in the cytoplasm and with some HSP60 in the mitochondria during electron microscopic immunohistochemistry. The signal sequence would be removed after protein import into the mitochondria and is not detectable by immunoblotting because of its low molecular mass. On the contrary, the cleavage and digestion of the signal sequence would not be performed simulta- neously with import of the protein into the mitochondria. However, in immunohistochemistry an anti-HSP60 signal sequence antibody reacted with both the signal sequence in the cytoplasm and mitochondria. In the present study, the purified cytoplasmic HSP60 inhibited thermal protein aggregation in vitro.Inthein vitro mitochondrial import reaction, the purified cytoplasmic HSP60 was imported into the mitochondria. Taken together, these results indicate that the mammalian HSP60 is localized in both the cytoplasm and the mitochondria in almost the same amounts. There are few reports concerning the import system of HSP60 into the mitochondria. In normal mammalian tissues, HSP60 is detected both in the cytoplasm and mitochondria. Newly synthesized HSP60 in the cytoplasm will be imported into the mitochondria under appropriate conditions. In the present study, we observed the import of the protein into the mitochondria of the water-restricted rat kidneys. The osmotic pressure increased in the rat kidney. In the kidney, there are some differences in the osmotic pressure in the cortex, medulla, and papilla. Among these three sections the papilla is most affected by water restriction. Although the HSP60 mRNA was not changed in the cortex and medulla of the kidney, HSP60 mRNA increased in the papilla of the kidneys of rats that had been water-restricted for 3 and 5 days. These data were also obtained during immunoblotting. The HSP60 in the cytoplasm and mito- chondria of the cortex and medulla did not change in their quantity or localization. However, the cytoplasmic HSP60 in the papilla decreased in response to water-restriction, and the mitochondrial HSP60 in the papilla was increased. Many proteins in the water-restricted rat kidneys were exposed to osmotic stress under these conditions and they became damaged. There are two speculations for the sorting of HSP60 into the mitochondria under conditions of water restriction: (a) some proteins in the cytoplasm of the water-restricted rat kidney’s papilla change their conformation and become aggregated. These proteins can be correctly folded by HSP70, which dissociates from Fig. 5. Signal sequence affinity column chromatography. (A) Rat liver cytoplasm was applied to the affinity column, and the binding proteins were eluted by 1 m M signal peptide. All samples were subjected to SDS/PAGE (13% polyacrylamide gel). Lane 1, rat liver cytoplasm; lane 2, proteins washed from the column; lane 3, proteins eluted by 1m M signal peptide; lane 4, molecular standard proteins. (B) The binding proteins were eluted from the affinity column with a linear gradient of signal peptide. The eluants were subjected to SDS/PAGE (10% polyacrylamide gel) followed by Coomassie Brilliant Blue staining. Rat liver cytoplasm was applied to the signal sequence affinity column, and the binding proteins were eluted by 1 m M signal peptide or 5 m M ATP. All samples were subjected to SDS/PAGE (9% poly- acrylamide gel) (C) and immunoblotting analysis using an antibody against HSP70 (D). Lane 1, Rat liver cytoplasm; lane 2, pass-through fraction from the column; lane 3, proteins washed from the column; lane 4, proteins eluted from the column by 1 m M signal peptide; lane 5, proteins eluted from the column by 5 m M ATP; lane 6, molecular standard proteins. 5936 H. Itoh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the signal sequence of HSP60 and plays a role as a molecular chaperon under these conditions. HSP60 can then be imported into the mitochondria, due to the free signal sequence of the protein; (b) some proteins in the mitochondria of the water-restricted rat kidney’s papilla change their conformation and become aggregated. To avoid these aggregated proteins, HSP60 will be imported into the mitochondria where it plays a role as a molecular chaperone. In the other sections of the water-restricted rat kidney the quantity and localization of HSP60 is not changed. These sections are either less- or are unaffected by the osmotic stress, and the quantity and localization of HSP60 in the cytoplasm and mitochondria of these sections are not changed even under these conditions. In the present study, HSP70 was bound to the signal sequence of the HSP60 affinity column, and HSP70 was dissociated from the column by the excess molar ratio of the HSP60 signal peptide or ATP. No other protein was found in the eluant from the affinity column. We confirmed the reverse experiment using an antibody against HSP70 in IgG-affinity column chromatography. We could observe the dissociation of HSP60 from the IgG column. These results indicated that HSP70, not MSF (mitochondrial import stimulation factor), is bound to the signal sequence of HSP60 near the mitochondria and that HSP60 is imported into the mitochondria when the signal sequence of HSP60 is exposed in the presence of ATP. We have shown here the import system of mammalian HSP60 into the mitochondria. Mammalian HSP60 is synthesized and localized stably in the cytoplasm, and the protein plays a role as a molecular chaperone or an immunophilin in the cytoplasm [12,13]. It has been reported that an unprocessed precursor of mitochondrial HSP60 stably existed in the yeast cytoplasm [24]. It has been shown that HSP60 associates with p21 ras [25] and that the protein is a major target for modification during S-(1,1,2,2,-tetrafluoroethyl)- L -cystein-induced nephrotoxici- ty [26]. Like those, HSP60 is located in the cytoplasm and has some physiological functions in the cytoplasm under physiological conditions. Very recently, it has been shown that cytosolic (nonmitochondrial) HSP60 forms a macro- molecular complex with Bax and Bak14. The complex formation with HSP60 may block the ability of Bax and Bak to effect apoptosis. These results suggest that the interactions of HSP60 with Bax and/or Bak regulate apoptosis. When cells or animals are exposed to a lethal environ- ment, HSP60 is quickly imported into the mitochondria under conditions of water restriction. HSP60 may play the role as a molecular chaperone in the mitochondria. The import mechanism of HSP60 into the mitochondria is mediated by the cytoplasmic HSP70. ACKNOWLEDGEMENTS We thank Dr. K. Nagata (Kyoto University) for his helpful comments on the manuscript. We thank Dr. D. J. Naylor (The University of Adelaide, Australia) for providing the rat Cpn10 (HSP10) expression vector (pRSC550-Cpn10). This work was supported in part by Grants- in-aid for Scientific Research (priority areas of molecular chaperone: 09276201, 10172201, and 11153201 to H. I., C2: 12670105 to H. I., C2: 14571011-00 to A.K., C2: 14570442 to M.O.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. REFERENCES 1. Hartl, F.U. (1996) Molecular chaperones in cellular protein fold- ing. Nature 381, 571–579. 2. Bukau, B. & Horwich, A.L. 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