©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mammalian 60-kDa Stress Protein (Chaperonin Homolog)
IDENTIFICATION, BIOCHEMICAL PROPERTIES, AND LOCALIZATION (*)

Hideaki Itoh (§) , Ryoji Kobayashi (4), Hideki Wakui (1), Atsushi Komatsuda (1), Hiroshi Ohtani (1), Akira B. Miura (1), Michiro Otaka (2), Osamu Masamune (2), Hideaki Andoh (3), Kenji Koyama (3), Yasuhiko Sato , Yohtalou Tashima

From the (1) Department of Biochemistry, the Third Department of Internal Medicine, the (2) First Department of Internal Medicine, and the (3) First Department of Surgery, Akita University School of Medicine, Akita 010, Japan and the (4) Department of Chemistry, Kagawa Medical School, Kagawa 761-07, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammalian chaperonin homolog (HSP60) was purified from porcine livers cytosol using a tandem ATP-Sepharose column and Mono Q column chromatography. A partial amino acid sequence (96 amino acid residues) of this protein was determined and coincided with those of human HSP60 with 96.9% homology, which was deduced from the nucleotide sequence of the cDNA. The sequence of the NH termini of the purified protein (5 amino acid residues) coincided with the signal sequence of HSP60. These facts led to the identification of the 60-kDa liver protein with the chaperonin homolog. Dihydrofolate reductase was able to form a stable complex with the liver chaperonin homolog. The liver chaperonin homolog was detected by at least five spots around pI = 5.6 on two-dimensional gel electrophoresis. Immunoblotting studies using an antibody against chaperonin homolog showed that the chaperonin homolog was localized in the cytosol, mitochondrial, and nuclear fractions of porcine liver. The chaperonin homolog was localized both in the mitochondria and cytoplasm of rat kidneys at the electron microscopic level. The chaperonin homolog in the cytosol, but not in the other subcellular fractions, was cross-reacted with an antibody against the synthetic peptide corresponding to the signal peptide of HSP60 as well as the purified chaperonin homolog on immunoblotting. These results suggested that the functional chaperonin homolog in the cytosol may be transported into the mitochondria and the protein may be processed to mitochondrial HSP60 in the organella.


INTRODUCTION

All living cells display a rapid molecular response to adverse environmental conditions, a phenomenon designated as the heat shock response. Because other kinds of stress show similar effects, this process can be considered as a general cellular response to metabolic disturbances. The most striking feature of the heat shock response is the induced synthesis of a set of proteins conserved during evolution, the heat shock proteins (HSPs)() or stress proteins (for reviews, see Refs. 1-3).

The stress proteins are usually classified on the basis of their approximate molecular masses and degrees of homology (for reviews, see Refs. 1-3). Most stress proteins are also synthesized constitutively in significant amounts under normal, nonstressed conditions. This raises the possibility that stress proteins play some important roles in the physiology of normal cells.

Many stress proteins, including members of the HSP70 and HSP60 families (molecular masses of 70 and 60 kDa, respectively), are constitutively expressed and fulfill essential functions as ``molecular chaperones'' under normal cellular conditions (4, 5) . The HSP60s, which are found in bacterial cytosol as well as in the mitochondria and chloroplast, are present in a highly self-associated form, double-ring complexes consisting of 14 60-kDa subunits (groEL) (6, 7) . These ``chaperonins'' interact with early intermediates in the protein folding pathway and mediate the acquisition of the native structure of newly synthesized proteins by releasing the substrate in an ATP-dependent process (8, 9) . The ATP hydrolytic activity of chaperonins is regulated by smaller co-chaperonins, a ring complex of seven 10-kDa subunits (groES) (10, 11) .

On the other hand, there are few reports on mammalian HSP60 because of the difficulty of purification of the protein. Guputa and co-workers (12, 13) originally raised an antibody against a 63-kDa mitochondrial protein (P1 protein) of Chinese hamster ovary cells, which was purified from excised protein spots in a large number of gel sheets of two-dimensional gel electrophoresis. They have recently isolated a complete cDNA encoding this protein from HL-60 cells (14) . The amino acid sequence deduced from the nucleotide sequence of the cDNA shows a homology to those of the bacterial and plant chaperonins and to the 65-kDa major antigen of mycobacterial 65-kDa stress protein (HSP65). Thus, the human P1 protein is now called human HSP60 or a chaperonin homolog (15) .

It has been shown that the degree of the sequence conservation between groEL and P1 protein (HSP60) is very similar; 50% of the amino acid residues are identical, and an additional 20% of the residues are conservative replacements (14) . HSP60 has a signal sequence (1-26 amino acid residues from the amino termini of HSP60). The protein may be transported into the mitochondria by a process similar to that of other imported mitochondrial proteins. HSP60 is converted into a mature form of lower molecular mass (58 kDa) in the mitochondria. The import of HSP60 into mitochondria is inhibited in the presence of an uncoupler, and also no import occurred when the amino-terminal presequence (signal sequence) is lacking (15) . Because of the above reasons, we distinguish HSP60 (chaperonin homolog) from mitochondrial HSP60 in this report.

Recently, Mizzen et al.(16) have reported the purification of mitochondrial HSP60 from HeLa cells using detergents and stated that the protein had no ATP-binding activity. We also have reported the purification of mitochondrial HSP60 using guanidine hydrochloride and observed that the protein had collagen-binding activity (17) . However, there is no report on the characteristics of mammalian HSP60. Several important physiological roles of the proposed HSP60 are raised. For example, Ikawa and Weinberg have reported the association of HSP60 with p21(18) .

The purification of mammalian HSP60 is essential to understanding its physiological functions. In the present study, we tried to purify and characterize HSP60 (chaperonin homolog) and will discuss its possible roles.


MATERIALS AND METHODS

ATP-Sepharose was prepared as described previously (19) . DE-52, Mono Q column, and ampholines were from Pharmacia Biotech Inc. Lysyl endopeptidase (EC 3.4.21.50) was from Wako Pure Chemical Industries (Osaka, Japan). GroEL was from Boehringer Mannheim GmbH (Germany), and anti-Yersinia enterocolitica HSP60 monoclonal antibody was from Wako Pure Chemical Industries (Osaka, Japan). Bovine liver dihydrofolate reductase (EC 1.5.1.3) was from Sigma. Fresh porcine livers were obtained from a local slaughterhouse and stored at -30 °C prior to use.

Purification of the 60-kDa Protein from Porcine Liver

Purification of the 60-kDa protein involved a slightly modified version of the purification method of HSP73 as described previously (19) . All operations were carried out at 0-4 °C. Frozen livers (about 800 g) were chopped and then homogenized with 3 volumes of buffer A (10 mM Tris-HCl, 15 mM 2-mercaptoethanol, 0.1% phenylmethylsulfonyl fluoride, pH 7.4). The homogenate was centrifuged at 20,000 g for 20 min. The supernatant was fractionated with ammonium sulfate added at a concentration of 27.7 g/100 ml. After the mixture was stirred for 30 min, the precipitates were discarded by centrifugation at 20,000 g for 10 min, and additional ammonium sulfate was added at 33.9 g/100 ml of the supernatant. The precipitates were collected by centrifugation, dissolved in buffer B (10 mM Tris-HCl and 15 mM 2-mercaptoethanol, pH 7.4), and dialyzed overnight against buffer B; the dialysis solution was changed several times. The dialysate was applied onto a DEAE-cellulose column (5 10 cm) pre-equilibrated in buffer B. After the column was washed with buffer C (10 mM Tris-HCl, pH 7.4), the proteins were eluted with 3 column-bed volumes of buffer C containing 0.15 M NaCl. The eluate fractions containing protein peaks were combined, and MgCl was added at a final concentration of 5 mM. The solution was applied onto a tandem ATP-Sepharose column (two columns were connected directly: the upper column, 2 7 cm; the lower column, 2 3 cm) pre-equilibrated with buffer D (10 mM Tris-HCl, and 5 mM MgCl, pH 7.4). After the columns were removed from each other, each column was washed with 3 column-bed volumes of buffer D containing 0.5 M NaCl and then washed with the same volumes of buffer D. The proteins were eluted with buffer D containing 3 mM ATP and fractionated for each column. If necessary, the elutant from the lower ATP-Sepharose column was applied onto a Mono Q column pre-equilibrated in buffer C and eluted with a linear gradient of 0-0.6 M NaCl in buffer C. The protein peak fractions were combined and stored at -80 °C.

Purification of Mitochondrial HSP60

Mitochondrial HSP60 was purified from porcine livers or porcine kidneys using guanidine hydrochloride as described previously (17) .

Gel Electrophoresis

SDS-PAGE was carried out by the procedure of Laemmli (20) using 7-10% polyacrylamide gel. Two-dimensional gel electrophoresis was performed as described by O'Farrell (21) . Samples were electrophoresed in the first dimension on an isoelectric focusing gel with a pH gradient of 5-7. Electrophoresis for the second dimension was performed on a 7% acrylamide slab gel with SDS. After electrophoresis, the gels were stained with 0.1% Coomassie Brilliant Blue R-250 in a mixture of 25% isopropyl alcohol and 10% acetic acid and destained with 10% isopropyl alcohol and 10% acetic acid.

Antibody Production

An antibody to the porcine 60-kDa liver protein was produced by intramuscular injection into a rabbit of 1 mg of the protein emulsified in complete Freund's adjuvant. Booster shots were given 3 times in the same manner as the original injection at 2-week intervals. The rabbit was bled 10 days after the last injection. Peptide corresponding to amino-acids 1-7 of the signal peptide of human HSP60 (MLRLPTV) was synthesized by solid phase techniques on an Applied Biosystems peptide synthesizer (Foster City, CA) model 433A with Fmoc (N-(9-fluorenyl)methoxycarbonyl) 1-hydroxybenzotriazole/N-methylpyrrolidone chemistry. After cleavage, peptide was purified by reverse-phase HPLC before use. The synthetic peptide (2.0 mg) was solved in 0.15 ml of phosphate-buffered saline (8.1 mM NaHPO, 1.5 mM KHPO, 2.7 mM KCl, 137 mM NaCl (pH 7.4)). The solution was mildly shaken with 0.3 ml of 50% (v/v) polyvinyl pyrolidone in phosphate-buffered saline for 2 h at room temperature. The conjugated solution, emulsified in complete Freund's adjuvant, was injected into a rabbit intramuscle. Booster shots were given 3 times in the same manner as the original injection at 2-week intervals. The rabbit was bled 10 days after the last injection.

The protocols for animal experimentation described in this paper were previously approved by the Animal Research Committee, Akita University School of Medicine; the ``Guidelines for Animal Experimentation'' of the University were completely adhered to in all subsequent animal experiments.

Immunodetection of 60-kDa Protein

Porcine livers were subcellularly fractionated as described previously (22) . All operations were carried out at 0-4 °C . Porcine livers were homogenized with buffer (10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 0.1 mM EDTA). After centrifugation at 7,000 g for 10 min, the precipitates (P1) and supernatant (S1) were treated by further centrifugation. The precipitates (P1) were dissolved in 0.25 M sucrose and centrifuged at 5,000 g for 10 min. The 5,000 g precipitate was used as the mitochondrial fraction. The supernatant (S1) was centrifuged at 54,000 g for 60 min, and the precipitate was used as the microsomal fraction. The 54,000 g supernatant was centrifuged at 105,000 g for 60 min. The 105,000 g supernatant was used as the cytosol. For the nuclear fraction, porcine livers were homogenized with 20 volumes of 2.2 M sucrose and centrifuged at 105,000 g for 60 min. The precipitate was used as the nuclear fraction. The purities of four subcellular fractions were checked by measuring marker enzymes or DNA, which localize in each fraction (cytosol, alcohol dehydrogenase (EC 1.1.1.1); mitochondrial fraction, succinate-cytochrome c reductase (EC 1.3.99.1); microsomal fraction, glucose-6-phosphatase (EC 2.4.1.22); nuclear fraction, DNA) and by using an electron microscope. Alcohol dehydrogenase, succinate-cytochrome c reductase, glucose-6-phosphatase, and DNA were determined as described by Lad and Leffert (23) , Bernath and Singer (24) , Nordlie and Arison (25) , and Anderson and Skagen (26) , respectively. Samples were developed on SDS-PAGE, electrophoretically transferred to a polyvinylidene difluoride membrane and processed as described by Towbin et al.(27) for immunoblotting. After the membranes were incubated with an anti 60-kDa liver protein antibody (diluted 1:1,000 in 7% skim milk), an antibody against the synthetic peptide corresponding to the signal sequence of HSP60 (diluted 1:200 in 7% skim milk) or anti-HSP60 monoclonal antibody (diluted 1: 20 in 7% skim milk), they were subsequently treated with horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad) (diluted 1:1,000 in 7% skim milk) or treated with horseradish peroxidase-conjugated anti-mouse IgG (Bio-Rad) (diluted 1:1,000 in 7% skim milk). Antigen-antibody complexes were visualized by reacting the bound peroxidase with 3,3`-diaminobenzidine and HO.

Amino Acid Sequence of 60-kDa Liver Protein

The purified 60-kDa liver protein or the 78-, 73-, 43-, and 30-kDa proteins, which were eluted from the ATP-Sepharose column, were electrophoresed on SDS-polyacrylamide gel, stained with 0.1% Coomassie Brilliant Blue R-250 in a mixture of 25% isopropyl alcohol, and these protein bands were excised. Using a lysyl endopeptidase, production and separation of peptides from the protein were carried out according to the method of Kawasaki et al.(28) . The peptides were purified by HPLC. A column of Wakopak (Wakosil 5C) was connected to an HPLC apparatus (Pharmacia LKB HPLC equipped with two model 2150 pumps, a model 2150 HPLC controller, and a model 2158 Uvicord S.D. UV detector was used for purification of the peptides). The peptide was applied onto the column pre-equilibrated with 0.1% trifluoroacetic acid and eluted with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min, and 0.5-ml fractions were collected. The purified peptides were detected at 206 nm. Because the NH terminus of the purified protein was blocked, a protein deblocking kit (Takara Shuzo, Kyoto, Japan) was used to determine the NH termini of the purified 60-kDa protein. The determination of the NH-terminal amino acid sequence of the purified peptide or deblocked protein was performed with a model 477A protein sequencer with an on-line 120A phenylthiohydantoin analyzer (Applied Biosystems, Foster City, CA).

DHFR Spectrophotometric Assay

DHFR enzyme assays were conducted in quartz cuvettes with a 1-cm path length according to the method of Viitanen et al.(29) . Enzyme activity was assayed in the direction of NADPH oxidation in the presence of dihydrofolate (HF). The reactions (final volume, 600 µl) were constructed in 1.5-ml microfuge tubes containing 0.05 M Tris-HCl, pH 7.4, 5 mM MgCl, 3.3 mM KCl, 10 mM dithiothreitol, and the purified 60-kDa protein (1.0 µM), in the presence or absence of HF (0.1 mM) and NADPH (0.1 mM). The reaction was initiated by the rapid addition of native DHFR or denatured DHFR by 5 M guanidine hydrochloride to a final concentration of 0.5 µM. After this addition, the reactants were immediately transferred to cuvettes, and A was monitored or the reactants were incubated for 5, 15, or 30 min prior to their supplementation with HF (0.1 mM) and NADPH (0.1 mM). After this addition, these reactants were immediately transferred to cuvettes, and A was monitored.

Electron Microscopic Immunohistochemistry

Electron microscopic immunohistochemistry of rat kidneys was performed as described previously (30) . Ultrathin sections of rat kidneys were cut with a diamond knife and mounted on gold grids. The sections were stained by the immunogold-silver staining method for electron microscopy using a silver enhancing kit (BioCell Research Laboratories). These sections were incubated with an antibody against the 60-kDa protein (200-fold dilution in buffer E: 10 mM phosphate buffer, pH 7.4, containing 2% NaCl and 0.1% Tween 20) for 18 h at room temperature. The sections were washed with buffer E and incubated with gold-labeled anti-rabbit IgG (Nanoprobes, NY; 400-fold dilution in buffer E) for 1 h at room temperature. After further washing with distilled water, the sections were incubated with the silver developer of the enhancing kit for approximately 10 min at room temperature. Sections were counter-stained with uranyl acetate and lead citrate.

Immunohistochemistry

Tissue fixation and immunoperoxidase staining were performed as described previously (31) . Briefly, tissue pieces were fixed with peroxidase lysine, 4% paraformaldehyde. Rat liver and pancreas were sliced (5 µm) in a cryostat. After blocking of endogenous peroxidase using 0.3% HO in methanol, the tissue sections were incubated with anti-60-kDa liver protein antibody (diluted 1:250 in 5% bovine serum albumin) for 12 h at 4 °C. The sections were then incubated with biotinylated anti-rabbit IgG (Vector Labs) for 40 min at room temperature, and avidin-biotin-peroxidase complex (Vector Labs) was applied for 40 min at room temperature. Sites of peroxidase activity were visualized with 0.02% 3,3`-diaminobenzidine-tetrahydrochloride containing 0.005% HO.

General Method

Proteins were measured by the method of Bradford (32) using bovine serum albumin as the standard. In this study, we used HSP73 prepared as described previously (19) and porcine liver GRP78 prepared by the method submitted for publication.


RESULTS

Purification of the Porcine 60-kDa Liver Protein

The liver 60-kDa protein was purified by the method described under ``Materials and Methods.'' In this method, tandem ATP-Sepharose column chromatography was employed. When the eluted fractions from DEAE-cellulose column were chromatographed on the upper ATP-Sepharose column, some proteins (subunit molecular mass of 78-, 73-, 68-, 60-, 43-, and 30-kDa proteins) were eluted from the column (Fig. 1). In these proteins, the 60-kDa protein was a minor protein. These proteins were electrophoresed on SDS-polyacrylamide gel (9% gel) and digested with lysyl endopeptidase, and the digests were purified by HPLC and subsequently sequenced by a protein sequencer. The 78-, 73-, 68-, and 43-kDa liver proteins were identical with GRP78, HSP73, and the NH-terminal fragments of HSP73, respectively (data not shown). On the other hand, the 60-kDa protein was eluted as a major protein from the lower ATP-Sepharose column with minor HSP73 (Fig. 2). The eluted protein pattern was quite different from the upper ATP-Sepharose column chromatography. After the Mono Q column chromatography, the final preparation gave only a single-band protein (60 kDa) by SDS-PAGE (Fig. 3). Four milligrams of the protein was obtained from 800 g of fresh porcine liver.


Figure 1: Tandem ATP-Sepharose column (the upper column) chromatography of the 60-kDa liver protein. The eluted fractions from the DEAE-cellulose column were chromatographed on a tandem ATP-Sepharose column. The elution was carried out with 3 mM ATP. A, elution pattern of protein from the upper ATP-Sepharose column. B, the eluted fractions were subjected to SDS-PAGE (9% gel) followed by Coomassie Brilliant Blue staining. Lane numbers correspond to the fraction numbers. LaneM, standard marker proteins: phosphorylase b (94 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa).




Figure 2: Tandem ATP-Sepharose column (the lower column) chromatography of the 60-kDa liver protein. The eluted fractions from the DEAE-cellulose column were chromatographed on a tandem ATP-Sepharose column. The elution was carried out with 3 mM ATP. A, elution pattern of protein from the lower ATP-Sepharose column. B, the eluted fractions were subjected to SDS-PAGE (9% gel) followed by Coomassie Brilliant Blue staining. Lane numbers correspond to the fraction numbers. LaneM, standard marker proteins.




Figure 3: SDS-PAGE of the 60-kDa liver protein. The purified 60-kDa liver protein was electrophoresed on 9% polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane1, the purified 60-kDa liver protein; lane2, standard marker proteins.



Amino Acid Sequence of the 60-kDa Liver Protein

Because the NH terminus of the purified 60-kDa liver protein was blocked, the intact NH terminus of the protein could not be sequenced. After the protein was digested with lysyl endopeptidase, the digests were purified using a C reverse-phase column, which was connected to an HPLC. Fig. 4 shows a peptide map obtained from the 60-kDa liver protein. The seven peptides (numbers 36, 43, 54, 61, 64, 68, and 93) were sequenced by a protein sequencer (Fig. 5A). Peptide 61 had contained three peptides. The obtained 91 amino acid residues from the seven peptides have 96.9% homology to human HL-60 HSP60, the amino acid sequence of which was deduced from the nucleotide sequence of the cDNA (14) . Deblocking of the blocked protein was performed, and the NH terminus of the protein was sequenced (Fig. 5A). The obtained 5 residues coincided with the signal sequence of human HL-60 HSP60 (14) . The homology of the partial amino acid sequences and the molecular mass led to the conclusion that the 60-kDa liver protein is the chaperonin homolog (HSP60). On the contrary, the intact NH terminus of the liver mitochondrial HSP60 could be sequenced (Fig. 5B). The signal sequence was lacking in the protein.


Figure 4: HPLC-fractionation of lysyl endopeptidase digests of the 60-kDa liver protein. Lysyl endopeptidase digests of the 60-kDa liver protein were separated by reverse phase chromatography on a C column with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. The purified peptides indicated in the panel (#36, #43, #54, #61, #64, #68, and #93) were sequenced by a peptide sequencer.




Figure 5: Sequence alignment of the 60-kDa liver protein and human HL-60 chaperonin homolog. The seven purified peptides of lysyl endopeptidase digests (#36, #43, #54, #61, #64, #68, and #93) or the NH terminus of the protein (A), and porcine liver mitochondrial HSP60 (B) were sequenced and compared with human HL-60 chaperonin homolog. Identical residues are denoted by a dash. Parentheses indicate the position of human chaperonin homolog.



Characterization of Chaperonin Homolog

To determine the isoelectric point of the chaperonin homolog, two-dimensional gel electrophoresis was carried out. The purified chaperonin homolog, bovine brain HSP73, and porcine liver GRP78 were co-electrophoresed, and the gel was stained with Coomassie Brilliant Blue (Fig. 6). The results showed microheterogeneity of the chaperonin homolog (five spots); the pI values of chaperonin homolog and HSP73 were approximately the same, 5.6 for chaperonin homolog and 5.8 for HSP73, but they were distinct from the value for GRP78, 5.2.


Figure 6: Two-dimensional gel electrophoresis of the 60-kDa liver protein. Purified 60-kDa liver protein, porcine liver GRP78, and bovine brain HSP73 were mixed and analyzed by two-dimensional gel electrophoresis. Isoelectric focusing (pH 5-7) was employed for the first dimension (basic end on the left, acidic end on the right) and SDS-PAGE (7% gel) for the second dimension. The gel was stained with Coomassie Brilliant Blue. Arrowsa, b, and c show the GRP78, HSP73, and the 60-kDa liver protein, respectively.



DHFR Can Interact with Chaperonin Homolog

When DHFR is preincubated, in the absence of a substrate, with a 2-fold molar excess of chaperonin homolog, a slow time-dependent loss of DHFR activity is subsequenced observed (Fig. 7, tracesb-e). DHFR ligands are unable to prevent unfolded DHFR from interacting with chaperonin homolog upon dilution from guanidine hydrochloride (Fig. 7, tracea). Native DHFR interacted with chaperonin homolog, but only in the absence of substrates. These results were approximately the same as those for groEL (29) .


Figure 7: Interaction between DHFR and the 60-kDa liver protein. Reactions were constructed in microfuge tubes containing the 60-kDa liver protein (1.0 µM), in the presence (tracesa and e) or absence (tracesb-d) of HF (0.1 mM) and NADPH (0.1 mM) as described under ``Materials and Methods.'' Reactions were initiated by the rapid addition of denatured DHFR (tracea) or native DHFR (tracesb-e). After this addition, the reactions shown in tracesa and e were immediately transferred to cuvettes, and A was monitored (t = 0 min). The remaining reactions were incubated for either 5 (traced), 15 (tracec), or 30 min (traceb) prior to their supplementation with HF (0.1 mM) and NADPH (0.1 mM). After this addition, the reactions shown in tracesa and e were immediately transferred to cuvettes, and A was monitored (t = 0 min).



Subcellular Localization of Chaperonin Homolog in Porcine Liver

The antibody against the purified liver chaperonin homolog was cross-reacted only with the 60-kDa protein in the liver crude extracts (Fig. 8B). The antibody is highly specific for the antigen. Porcine livers were subcellularly fractionated as described previously (22) , and the purities of four subcellularly fractions were checked by measuring marker enzymes or DNA. Each sample was analyzed by SDS-PAGE and the immunoblotting method using anti porcine liver chaperonin homolog antibody. Chaperonin homolog was detected not only in the mitochondrial fraction but also in the nuclear and cytosol fractions. The present experiments did not confirm its presence in the microsomal fraction (Fig. 8B, lanes1-4). The antibody against synthetic peptide corresponding to the signal peptide of HSP60 was cross-reacted with the 60-kDa protein in the cytosol and the purified chaperonin homolog (Fig. 8C). Although the immunoreactivity was extremely weak, the purified liver chaperonin homolog and the 60-kDa protein in subcellular fractions were detected on the immunoblotting method using an anti-Y. enterocolitica HSP60 monoclonal antibody, which cross-reacts with groEL, various bacteria, and mammalian HSP60 (Fig. 8D). These results indicated that mammalian chaperonin homolog exists both in the mitochondria and cytosol.


Figure 8: Subcellular localization of the 60-kDa liver protein. Porcine liver was subcellularly fractionated, and the fractions were electrophoresed on 9% SDS-polyacrylamide gels, which were stained with Coomassie Brilliant Blue (A), by immunoblotting with an antibody against 60-kDa liver protein (B), by immunoblotting using an antibody against synthetic peptide corresponding to the signal sequence of HSP60 (C), or by immunoblotting using a monoclonal antibody against Y. enterocolitica HSP60 (D). Lanes1, cytosol fraction; lanes2, microsomal fraction; lanes3, mitochondrial fraction; lanes4, nuclear fraction; lanes5, purified 60-kDa liver protein; lanes6, standard marker proteins.



Histochemistry of Chaperonin Homolog in the Rat Liver and Rat Kidney

To elucidate the physiological roles of chaperonin homolog, the localization of chaperonin homolog in the normal rat liver was studied using the antiserum against the porcine liver chaperonin homolog (Fig. 9A). This antibody was cross-reacted with from human liver chaperonin homolog to groEL as well as porcine liver chaperonin homolog in the immunoblotting method (data not shown). In normal rat livers, chaperonin homolog was detected both in the cytoplasm and nucleus (Fig. 9A). To determine the localization of chaperonin homolog in the kidneys, electron microscopic immuno-histochemistry was performed. Chaperonin homolog was detected both in the mitochondria and cytoplasm (Fig. 9B). Based on the result shown in Fig. 8, the positive reactions in the cytoplasm are responsible for the presence of chaperonin homolog in the mitochondrial and cytosol fractions.


Figure 9: Immunohistochemical localization of the 60-kDa protein in rat liver and rat kidney. Thin sections of rat liver were stained with anti-60-kDa liver protein (A). Rat kidney sections were stained by the immunogold-silver staining method using antiserum against porcine liver 60-kDa protein (B). M, mitochondria; C, cytoplasm. Arrowhead in the panelB indicates localization of the 60-kDa protein. Bar, 1 µm.




DISCUSSION

We have identified and characterized constitutively expressed stress protein, chaperonin homolog, in porcine liver. The following evidence allows us to conclude that the 60-kDa protein purified from the livers is identical to chaperonin homolog: (i) subunit molecular mass of 60 kDa on SDS-PAGE; (ii) high homology (96.9%) of partial amino acid sequences (96 residues) between the liver protein and human HL-60 chaperonin homolog sequence of which was deduced from the nucleotide sequence of the cDNA; (iii) the sequence (5 residues) was coincided with the signal sequence of human HL-60 HSP60; (iv) DHFR can form a stable complex with the 60-kDa protein; (v) the liver protein was cross-reacted with the antibody against synthetic peptide corresponding to the signal peptide of HSP60.

For the purification of chaperonin homolog, tandem ATP-Sepharose column chromatography was very effective. It was interesting that the protein was eluted as a major protein from the lower ATP-Sepharose column, in spite of the elution of chaperonin homolog from the upper ATP-Sepharose column as a minor protein; the protein was rather selectively retained in the lower ATP-Sepharose column. These phenomena were observed in the purification of the protein from bovine brain. The reason why chaperonin homolog binds selectively to the lower column is obscure. We speculate that some proteins in the crude fractions might interfere with the binding of chaperonin homolog in the case of the upper ATP-Sepharose column. We are searching for such interesting molecules.

Mizzen et al.(16) also reported the low affinity of HeLa cell mitochondrial HSP60 and the high affinity of HSP72 for ATP-Sepharose (16) . They used Triton X-100 and sodium deoxycholate in the extraction buffer. The detergents may reduce the binding affinity of mitochondrial HSP60 to the gel. The present purification method is simple and highly reproducible, and its recovery is high. The method was available for chaperonin homolog from various mammalian organs including brains.

Chaperonin homolog showed a pI value of 5.6 on two-dimensional gel electrophoresis. The microheterogeneity of chaperonin homolog might be dependent on phosphorylation or charge heterogeneity. Native DHFR can form a stable complex with chaperonin homolog as those for groEL (29) . However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrate. As a result, most of the native enzyme can eventually be sequestered on the chaperonin in a native form. This might imply that chaperonin homolog can recognize certain protein species that possess considerable secondary and tertiary structure. The biochemical properties (molecular mass, pI, affinity for ATP-Sepharose, and chaperonin activity) of chaperonin homolog were very similar to those of bacterial groEL protein.

We obtained the monospecific polyclonal antibody against porcine chaperonin homolog. The antibody was cross-reacted with chaperonin homologs from human to E. coli (GroEL). Chaperonin homolog was detected in the cytosol and nuclear fractions as well as in the mitochondrial fraction on immunoblotting method. The data were supported by immunoblotting using an antibody against the synthetic peptide corresponding to the signal sequence of HSP60 and an anti-Y. enterocolitica HSP60 monoclonal antibody. The concentration of chaperonin homolog in the cytosol fraction was almost the same as that in the mitochondrial fraction and less in the nuclear fraction. Chaperonin homolog was also detected both in the cytoplasm and mitochondria on electron microscopic immunohistochemistry. These results suggested that chaperonin homolog, which may be induced in the cytosol, may be transported into mitochondria, and the protein may be converted into a mature form (mitochondrial HSP60) in the organella. HSP60 has no typical nuclear-localization signal like a PKKKRK. However, it was detected in the nuclear of normal rat liver in the present study. The mechanism of transferring into the nuclei is under experiment. It was reported that a small fraction of HSP60 is located in the cell membranes (33) or localized in the nucleus of a fish cell line (34) . These results seem to suggest that HSP60 plays some important roles not only in the mitochondria but also in the cytosol, nuclei, and cell membranes.

In the present report, we present evidence that mammalian chaperonin homolog possesses an ATP binding activity and chaperonin activity and localizes in nuclear, mitochondria, and cytosol. These findings represent the first demonstration of characterization of a mammalian chaperonin homolog and may help understand its function in normal and stressed cells, especially in intracellular protein folding and sorting.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Akita University School of Medicine, 1-1-1 Hondo, Akita City, 010 Japan. Tel.: 81-188-33-1166; Fax: 81-188-36-2606.

The abbreviations used are: HSP, heat shock protein; HSP60 and HSP73, heat shock proteins with subunit molecular mass of 60 kDa and 73 kDa; GRP78, glucose-regulated protein with subunit molecular mass of 78 kDa; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; DHFR, dihydrofolate reductase; HF, dihydrofolate; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Yuko Itoh for assistance in preparing the manuscript and Dr. Kazuhiro Nagata for helpful comments on the manuscript.


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