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

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

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].

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

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

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

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.

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

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

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

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

(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

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.
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