From the
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
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)
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;
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
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.
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.
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.
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.
We thank Yuko Itoh for assistance in preparing the
manuscript and Dr. Kazuhiro Nagata for helpful comments on the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
or stress
proteins (for reviews, see Refs. 1-3).
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.
(18) .
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
KH
PO
, 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.
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 H
O
.
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
H
F (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 H
F
(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%
H
O
.
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.
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
H
F (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.
F, dihydrofolate; HPLC,
high performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.