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The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay.. immunosuppressant mizoribine is cyt

Mammalian HSP60 is quickly sorted into the mitochondria

under conditions of dehydration

Hideaki Itoh1, Atsushi Komatsuda2, Hiroshi Ohtani2, Hideki Wakui2, Hirokazu Imai2, Ken-ichi Sawada2, Michiro Otaka3, Masahito Ogura1, Akira Suzuki1and Fumio Hamada4

1

Department of Biochemistry,2Department of Third Internal Medicine, and3Department of First Internal Medicine,

Akita University School of Medicine, Akita City, Japan;4Department 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 mMATP 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 mitochondmitochond-rial 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)

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] HSP60 may play a key role in

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

M A T E R I A L S A N D M E T H O D S

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

sequenceofhumanHSP60(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

Guidance for Animal Experimentation 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 HSP60in vitro

The purified cytoplasmic and mitochondrial HSP60 were

labelled with125I 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 mMMgCl2/ATP in 10 mMTris/HCl pH 7.4 for 60 min

at 37C 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 mMTris/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 43C was monitored as described [19] To monitor the thermal unfolding/aggregation, the CS concentration was 0.075 lMin 40 mMHepes buffer pH 7.4 in the presence or absence of purified porcine HSP60 (0.075 lM), recombinant rat HSP10 (0.075 lM), and ATP/MgCl2(5 mM) 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 mM each of the four dNTPs in 50 mMTris, pH 8.3, 75 mMKCl,

3 mM MgCl2 and 10 mM dithiothreitol for 1 h at 42C 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 lMeach 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

Subcellular fractionations of rat livers or

water-restricted rat kidneys

All operations were carried out at 0–4C The livers

were homogenized with buffer (10 mM Tris/HCl, pH 7.4,

0.25M sucrose, 0.1 mM 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 mM Tris/HCl, pH 7.4, 0.25M sucrose,

0.1 mM 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 mM Tris/

HCl, pH 7.4, 0.25M sucrose and 0.1 mM 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 mM Tris/HCl pH 7.4 and washed with 10 column

vols of the buffer containing 0.5M NaCl After washing

the column, binding proteins were eluted from the

column with the 0.1, 1 and 5 mM signal peptide of

HSP60 or 5 mM 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

R E S U L T S

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 (Mr¼ 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

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 4C or at 37 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),

an anti-CS Ig (J), anti-HSP90 Ig (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 I and incu -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.

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

43C [20,23] As shown in Fig 3, spontaneous aggregation

occurred at 43C 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 separawater-restric-ted 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.

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 mMATP and the same

70-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

D I S C U S S I O N

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 In the in 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

1 m 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.

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 p21ras [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

A C K N O W L E D G E M E N T S

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.

R E F E R E N C E S

1 Hartl, F.U (1996) Molecular chaperones in cellular protein fold-ing Nature 381, 571–579.

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