* Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 862, Japan; and Institut für
Biochemie und Molekularbiologie, D-79104 Freiburg, Germany
DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.
THE 70K heat shock protein (hsp70)1 family is a
group of molecular chaperones which mediates protein folding and targeting (reviewed in Bukau et al.,
1996 Three DnaJ homologues have been identified in human
cytosol: dj1 (hsp40/hdj-1) (Ohtsuka, 1993 Both dj1 and hsj1 contain a J-domain and G/F-domain,
but lack a cysteine-rich region and a farnesylation motif.
These structural features are similar to those of yeast Sis1p,
another member of the DnaJ family (Caplan and Douglas,
1991 To investigate the roles of hsc70 and the two cytosolic
DnaJ homologues dj1 and dj2 of mammals, we developed
systems of depletion-readdition of these chaperones. We
found that both hsc70 and dj2 are required for efficient
mitochondrial import of preornithine transcarbamylase (pOTC) during its synthesis. We also tested our depletion-
readdition systems to study possible differential roles of
these DnaJ homologues in folding of chemically denatured luciferase. Unexpectedly, dj2, but not dj1, facilitated
productive folding in cooperation with hsc70. DnaJ could
replace dj2 both in mitochondrial protein import and in
protein folding. Thus, the cytosolic chaperone system is highly conserved: DnaK-DnaJ-GrpE in bacteria, Ssa1-
4p-Ydj1p in yeast, and hsc70-dj2 in mammals.
Materials
The nuclease-treated rabbit reticulocyte lysate and the luciferase assay
system were purchased from Promega Corp. (Madison, WI). [35S]Pro-mixTM (>37 TBq/mmol [35S]methionine) was purchased from Amersham
Corp. (Arlington Heights, IL). Firefly luciferase was purchased from
Sigma Chemical Co. (St. Louis, MO).
Purification of Chaperones
Mouse hsc70 was purified from Ehrlich ascites fluid by ATP-agarose column chromatography and Superdex gel filtration column chromatography.
Human dj2 was expressed using the insect/baculovirus system (BaculoGold Transfection kit; PharMingen, San Diego, CA). The DNA fragment
of histidine-tagged human dj2 was excised from a plasmid encoding the
MBP-His-dj2 fusion protein (Kanazawa et al., 1997 Antibodies
Rat 1B5 mAb was purified from rat ascites fluid as described in Terada
et al. (1995) Immunoblot Analysis
Proteins were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Clear blot-P; Atto Co., Tokyo, Japan). The
membranes were probed with 1B5 mAb (2 µg IgG/ml) for detection of
hsc70, anti-human dj2 antiserum (1/2,500 dilution) for detection of dj2,
and anti-human dj1 antiserum (1/2,500 dilution) for detection of dj1 and
the chaperone polypeptides were identified using the biotin/avidin system
(ABC kit; Vector Laboratories, Burlingame, CA) and a chemiluminescence (ECL; Amersham).
Depletion of Chaperones from Rabbit
Reticulocyte Lysate
Depletion of hsc70 from rabbit reticulocyte lysates was done by treatment
with 1B5 antibody-resin (Terada et al., 1995 Import of In Vitro-synthesized pOTC into
Isolated Mitochondria
Translation of rat pOTC mRNA was performed in vitro in a rabbit reticulocyte lysate depleted for each chaperone. Where indicated, purified chaperones were added to the depleted lysates before translation. The import
mixture (50 µl) containing 5.0 µl of the lysate and 35S-labeled pOTC (2-23
KBq) was incubated with isolated rat liver mitochondria (100 µg of protein) at 25°C, as described (Terada et al., 1996 Refolding of Chemically Denatured Luciferase
Firefly luciferase (0.5 mg/ml, 8.1 µM) was denatured in buffer A (25 mM
Hepes-KOH, pH 7.2, 50 mM potassium acetate, 5 mM dithiothreitol) containing 6 M guanidine hydrochloride. The solution was incubated at 25°C
for 60 min. The denatured luciferase was placed on ice and diluted 1:40 in
buffer A. Then, 2.0 µl of diluted luciferase was added to 48 µl of refolding
buffer (28 mM Hepes-KOH, pH 7.6, 120 mM potassium acetate, 1.2 mM
magnesium acetate, 2.2 mM dithiothreitol, 1 mM ATP, 8.8 mM creatine
phosphate, 2 µg creatine kinase, 50 µM antipain, and 50 µM leupeptin)
containing 15 µl of lysate and/or chaperones where indicated. Refolding
was started by incubating at 25°C. At indicated times, 1.0 µl was withdrawn from the reaction and added to 50 µl of a luciferase assay solution
(Promega), and light production was immediately monitored for 12 s in a
luminometer (TD-20/20; Turner Designs, Sunnyvale, CA). In all experiments, the enzyme activity was expressed as a percent of that of the native
enzyme measured after dilution with buffer A containing 1 mg/ml bovine
serum albumin.
Intracellular Concentration of Cytosolic Chaperones
We purified chaperones including mouse hsc70, human recombinant histidine-tagged dj2 (His-dj2) and dj1 (His-dj1),
and Escherichia coli DnaJ (Fig. 1 a). hsc70 migrated as a protein of 73K in SDS-PAGE (lane 1). His-dj2 expressed in
Sf9 insect cells gave 49K and 47K polypeptides (lane 2). We
have shown that human dj2 is subject to farnesylation at
Cys-394 and that the farnesylated form migrates faster than
the unfarnesylated form on SDS-polyacrylamide gel (Kanazawa et al., 1997
We then examined intracellular concentrations of the
three cytosolic chaperones in rabbit reticulocytes, human
hepatoma HepG2 cells, monkey kidney COS-7 cells, and
rat liver (Fig. 1 b). Assuming that the cytosolic protein
concentration is 200 mg/ml, hsc70 concentration in HepG2
cells was estimated to be about 2.0 mg/ml of cytosol (30 µM, 1.0% of total cytosolic protein). Assuming that this
antibody against Chinese hamster hsc70 reacted equally
with the human, monkey, mouse, rat, and rabbit hsc70s,
the concentrations in COS-7 cells and rat liver were similar to that in HepG2 cells, whereas that in rabbit reticulocytes was much lower (0.31 mg/ml, 4.2 µM, 0.15% of total
protein). These events may reflect the fact that reticulocytes are highly specialized cells containing high contents of hemoglobin, that is ~90% of total protein in reticulocyte lysates. When an anti-mouse hsp70 antiserum that is
highly cross-reactive with hsp70s of other mammals was
used for immunodetection, extracts of HepG2 and COS-7
cells gave weak signals of 72K, whereas rabbit reticulocyte
lysate and rat liver cytosol gave no signal (data not shown).
The intracellular concentrations of dj2s in HepG2 cells,
COS-7 cells and rat liver were estimated to be 0.20-0.25
mg/ml (4.3-5.4 µM, 0.08-0.10% of total protein) and that
in the reticulocyte lysate was estimated to be 10 µg/ml
(0.22 µM, 0.05% of total protein). In addition to the dj2-immunoreactive polypeptide of 46K, another immunoreactive polypeptide of 49K was observed in the reticulocyte lysate, the nature of which is unknown.
Endogenous dj1s of the tested tissue and cells were detected as 40K proteins. The concentration was highest in
HepG2 cells (0.20 mg/ml, 5.0 µM, 0.10% of total protein),
that in the COS-7 cells was 80 µg/ml (2.0 µM, 0.04% of total protein), that in rat liver cytosol was 20 µg/ml (0.50 µM, 0.01% of total protein), and that in rabbit reticulocyte
lysate was 6.0 µg/ml (0.15 µM, 0.03% of total protein).
These values agree well with documented data (Frydman
et al., 1994 Development of Chaperone
Depletion-Readdition Systems
To investigate the effects of these chaperones on the import of pOTC into mammalian mitochondria, we developed a system of chaperone depletion from the reticulocyte lysate. We also tested this system to examine the roles
of chaperone(s) in the folding of a model protein luciferase. Antibody-coupled resins were prepared for each
of the chaperones. Mock-treated lysate prepared with nonimmune rabbit IgG-coupled resin or untreated lysate (two
times diluted) was used as controls for all experiments. Depletion of the chaperones was monitored by immunodetection (Fig. 1 c).
Depletion of hsc70 from the lysate was efficient and always exceeded 90% (Fig. 1 c, lane 2). The procedure of
hsc70 depletion led to a partial reduction of dj2 (by
~50%), whereas it caused a marked reduction of dj1 (by
~85%). These results indicate that most dj1, but not dj2, is
tightly associated with hsc70 in rabbit reticulocyte lysate.
These findings agree with those of Yamane et al. (1995) Effect of dj2 Depletion on pOTC Import
into Mitochondria
To investigate the roles of cytosolic chaperones in mitochondrial protein import, we synthesized rat pOTC in the
untreated or the chaperone(s)-depleted reticulocyte lysate
and import assays were done. When hsc70 (and concomitantly dj1) was depleted from the lysate, a decrease in
pOTC synthesis was observed (Terada et al., 1995
When dj1-depleted lysate was tested for pOTC import,
little decrease was observed (Fig. 2 c). Readdition of His-dj1 to the dj1-depleted lysate had little effect on import.
These results are consistent with those obtained for hsc70-depletion where dj1-depletion occurred concomitantly and
the import restoration was achieved by the readdition of
only hsc70. Efficiency of pOTC translation in the dj1-
depleted lysate was slightly lower than that in the mock-depleted lysate.
We next examined the effects of dj2-depletion. Contrary
to the effect of hsc70-depletion on translation efficiency,
dj2-depletion led to only a slight decrease. Mitochondrial
import of pOTC synthesized in the dj2-depleted lysate decreased to one-third that of the precursor synthesized in
the mock-depleted lysate (Fig. 2 b). This finding is in accord with our previous inhibition studies using anti-dj2 antibody (Kanazawa et al., 1997 Bacterial DnaJ is highly homologous to dj2, but lacks
farnesyl modification in its COOH terminus. We asked
whether DnaJ could replace dj2 in pOTC import into mitochondria. The pOTC import was nearly completely recovered when DnaJ was readded to the dj2-depleted lysate (data not shown). Thus, farnesyl modification is not
essential for the members of cytosolic DnaJ family in mitochondrial protein import, though it may be required for
functionally active dj2 in the context of its structural basis.
Facilitation of Refolding of Chemically Denatured
Luciferase by hsc70 and dj2
We next examined the roles of DnaJ homologues in folding of cytosolic proteins. dj1 (hdj-1) has been reported to
be the DnaJ homologue promoting folding of chemically
denatured luciferase (Freeman et al., 1995 Chemically denatured luciferase when incubated alone
at 25°C was not efficiently refolded into the enzymatically
active form (Fig. 3 a). On the other hand, it was efficiently
refolded in the untreated reticulocyte lysate. Refolding
of luciferase proceeded with time and reached a plateau
within 60 min. 87% of the enzyme activity was recovered
in 90 min. These results are in good accord with the documented data (Nimmesgern and Hartl, 1993
We next examined the effect of dj2-depletion on refolding activity of the lysate. Surprisingly, dj2-depletion caused a
dramatic decrease in refolding (Fig. 3 b). Requirement of
dj2 was further confirmed in readdition experiments.
When 0.2 µM His-dj2 was readded to the dj2-depleted lysate, an almost complete recovery of refolding was observed. Restoration of the refolding activity depended on the amount of added His-dj2 (Fig. 3 d). Thus, dj2 is required for productive folding of denatured luciferase. The
concentration of His-dj2 required for the maximal restoration was similar to that in the reticulocyte lysate and was
much lower than those in tissues and cultured cells.
To verify the effect of dj1, refolding of luciferase was
monitored in the dj1-depleted lysate. However, no significant
effect was observed regardless of whether or not dj1 was
present. When His-dj1 was readded to the dj1-depleted lysate,
a slight reduction in the refolding reaction occurred. Thus,
it is dj2, and not dj1, that cooperates with hsc70 to promote
productive folding of chemically denatured luciferase.
Effect of DnaJ on Luciferase Refolding
Since bacterial DnaJ could replace dj2 in mitochondrial protein import, we asked if DnaJ could replace dj2 in luciferase refolding. In combination with yeast cytosolic hsp70
Ssa1p, DnaJ was shown to refold luciferase efficiently (Levy
et al., 1995 Refolding of Luciferase by Purified Chaperones
Refolding of luciferase by these chaperones was studied in
greater detail using purified components. As shown in Fig.
4 a, luciferase refolding was somewhat facilitated by hsc70,
His-dj2, and His-dj1. However, bovine serum albumin at a
similar concentration was also effective. Therefore, the facilitation effects by these chaperones do not appear to be
due to their specific chaperone activities. On the other hand,
luciferase activity could be recovered by the addition of either His-dj2 or DnaJ together with hsc70. The addition of
His-dj1 together with hsc70 had no significant effect.
Kinetics of luciferase refolding by His-dj2 or DnaJ plus
hsc70 differed from that in the lysate (Fig. 4 b, see also Fig.
3 a). Refolding of luciferase was slow for the first 10 min
and then proceeded linearly up to 60 min. The reason for
the difference in refolding kinetics is unknown. However,
it is possible that other component(s) in the lysate supported folding activity of the hsc70-dj2 chaperone system.
This component may be another chaperone system(s) that
maintains denatured proteins in a folding-competent state(s)
and/or a regulatory element(s) that modulates folding activity of the hsc70-dj2 system.
Thus, it is obvious that hsc70 and dj2 constitute a mammalian chaperone system promoting mitochondrial import
of pOTC and the productive folding of luciferase. dj1 was
not effective in our studies, but it may play a role under
special conditions such as heat-stress. Minami et al. (1996) We found that dj2 among the mammalian DnaJ homologues participates in mitochondrial protein import and in
productive protein folding as an indispensable partner
chaperone of hsc70. Alignment of the sequences of DnaJ,
Ydj1p, and dj2 also suggest their functional similarity. Furthermore, functional replacement of mammalian dj2 (this
study) or yeast Ydj1p (Levy et al., 1995 We tested possible chaperoning activity of dj1 in mitochondrial protein import and in protein folding. However,
immunodepletion of dj1 from the rabbit reticulocyte lysate
did not affect refolding of denatured luciferase. Furthermore, purified His-dj1 in combination of hsc70 could not
support luciferase refolding. One reported refolding of luciferase by hdj-1(dj1)-hsc70 (Freeman et al., 1995 We previously showed that the requirement of hsc70 for
mitochondrial protein import varies among precursor proteins (Terada et al., 1996 It is striking that hsc70 and dj2 promote both folding of
a denatured protein and import of a protein into mitochondria. A similar enigma was presented in yeast and E. coli. In yeast, Ssa1-2p-Ydj1p promotes both protein folding and protein import into mitochondria and endoplasmic
reticulum (Cyr et al., 1994 Ydj1p was initially identified as a component necessary
for post-translational protein import into mitochondria and
endoplasmic reticulum (Caplan and Douglas, 1991; Hartl, 1996
; Rassow et al., 1997
). Members of the
hsp70 family usually require partner proteins for specifying their functions in distinct cellular compartments of eukaryotic cells (Rassow et al., 1995
). An essential and ubiquitous group of these partner proteins for hsp70 family is
the DnaJ family (Cyr et al., 1994
).
; Raabe and Manley, 1991
), dj2 (HSDJ/hdj-2) (Chellaiah et al., 1993
; Oh et al.,
1993
), and hsj1 (Cheetham et al., 1992
). The structure of
dj2 has the closest similarity to those of bacterial DnaJ and
yeast Ydj1p, Mdj1p, and Scj1p (Cyr et al., 1994
). These
members of the DnaJ subfamily have the J-domain, G/F-domain, and the cysteine-rich region. The cysteine-rich region of DnaJ coordinates two zinc atoms and is important
for binding to chemically denatured luciferase (Szabo et al.,
1996
). Ydj1p and dj2 also have the CaaX prenylation motif at their COOH termini and undergo farnesyl modification,
posttranslationally (Caplan et al., 1992a
; Kanazawa et al.,
1997
). Although the functional significance of this modification of dj2 is unknown, studies on Ydj1p revealed temperature-sensitive defects in protein import into mitochondria and endoplasmic reticulum with the lack of
farnesylation (Caplan et al., 1992b
).
; Luke et al., 1991
). Sis1p is localized mainly in the cytosol and partially in the nucleus and is essential for viability (Luke et al., 1991
). It also associates with ribosomes
and promotes the initiation of translation (Zhong and
Arndt, 1993
). The intracellular localization of human dj1 is
similar to that of yeast Sis1p (Hattori et al., 1993
). dj1 also
associates with ribosomes and is engaged in folding of nascent polypeptides (Frydman et al., 1994
). A protein refolding activity of dj1 was also reported (Freeman and
Morimoto, 1996
; Freeman et al., 1995
). hsj1 is preferentially expressed in neuronal tissues (Cheetham et al., 1992
),
and inhibits heat shock cognate 70 protein (hsc70)-catalyzed clathrin uncoating (Cheetham et al., 1996
).
Materials and Methods
), inserted into a
pVL1392 transfer vector, as specified by the manufacturer. Sf9 cells in suspension were infected with the recombinant virus and cultured for 72 h.
Farnesylation of His-dj2 was enhanced by exogenously adding a mevalonate precursor, mevalonolactone (Sigma), to the medium, as described
for the expression of small G proteins (Takai et al., 1995
). His-dj2-expressing Sf9 cells were lysed and His-dj2 was purified with a nickel chelate affinity column. Details of the expression and purification will be described
elsewhere. Histidine-tagged human dj1 was expressed in Escherichia coli
and purified as described (Minami et al., 1996
). In brief, pQE-9/Hsp40
plasmid was transformed into M15[pREP4] cells and grown at 30°C. After
4-h induction with 0.1 mM IPTG, cells were lysed and loaded onto Ni2+-NTA Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ) column. The column was washed with 60 mM imidazole and then proteins
were eluted with 1 M imidazole. Peak fractions of His-dj1 was dialyzed against 100 mM potassium phosphate buffer (pH 7.6) and loaded onto hydroxyapatite HTP column (Bio-Rad Labs., Richmond, CA). His-dj1 was
eluted with a linear gradient of 100-500 mM potassium phosphate buffer
(pH 7.6). The purified His-dj1 was concentrated by ultrafiltration.
. Anti-human dj2 antibody was that described previously
(Kanazawa et al., 1997
). Anti-dj1 antibody was raised in a rabbit by injecting purified histidine-tagged human dj1.
, 1996
). Depletion of other
chaperones was done with protein A-Sepharose 4FF beads (Pharmacia)
cross-linked to IgG from either nonimmune serum (mock-depleted), anti-dj2 serum (dj2-depleted) or anti-dj1 serum (dj1-depleted) by dimethylpimelimidate (Pierce Chemical Co., Rockford, IL) as described (Harlow
and Lane, 1988
). The antibody-resins were equilibrated with 20 mM
Hepes-KOH and 120 mM potassium acetate (pH 7.6) and an equal volume of reticulocyte lysate was added to the pelleted resins. The mixtures
were incubated at 4°C for 60 min with occasional agitation, and the resins
were removed by centrifugation.
). The reaction was stopped
by adding ice-cold mitochondria isolation buffer containing 0.1 mM dinitrophenol (Terada et al., 1995
). The mitochondria were reisolated and
subjected to SDS-PAGE. The radioactive polypeptides were visualized by
fluorography and quantitated using an imaging plate analyzer (FUJIX
BAS2000; Fuji Photo Film Co., Tokyo, Japan).
Results
). These two polypeptides also proved to be dj2 by immunoblot analysis. The ratio of unfarnesylated form to farnesylated one was ~1:2 (Fig. 1 b, middle,
lanes 5-9), but this ratio varied from one preparation to
another. When His-dj2 expressing insect cells were cultured in the absence of a mevalonate precursor, farnesylation was much decreased (data not shown). On the other
hand, human dj1 contains no prenylation motif, and a single 42K polypeptide was observed (Fig. 1 a, lane 3). E. coli DnaJ migrated as a 41K polypeptide (lane 4).
Fig. 1.
Intracellular concentrations of
chaperones and depletion for chaperones
of rabbit reticulocyte lysate. (a) Purified
chaperones (0.5 µg each) were analyzed
by SDS-PAGE. Lane 1, hsc70; lane 2, His-dj2; lane 3, His-dj1; lane 4, DnaJ. (b)
Immunoblot analysis of hsc70 (top), dj2
(middle), and dj1 (bottom) in rabbit reticulocyte lysate (lane 1, 20 µg protein),
HepG2 cell extract (lane 2, 2 µg protein),
COS-7 cell extract (lane 3, 2 µg protein),
and rat liver cytosol (lane 4, 2 µg protein).
Purified chaperones were used as standards (lanes 5-9): top, mouse hsc70 (8, 16, 31, 63 and 130 ng); middle, baculovirus-
expressed human His-dj2 (0.63, 1.3, 2.5, 5.0, and 10 ng); bottom, E. coli-expressed human His-dj1 (0.060, 0.13, 0.25, 0.50, and 1.0 ng).
Note that standard dj2 and dj1 had histidine tags and migrated more slowly than the endogenous chaperones in 8% SDS-PAGE. The
reason for the presence of immunoreactive 49K band in rabbit reticulocyte lysate is unknown (middle, lane 1). Human recombinant
hsc70 (a gift from N. Imamoto and Y. Yoneda) and mouse hsc70 gave signals of similar intensities. (c) Immunodepletion was performed
with antibody-coupled Sepharose resins as described in Materials and Methods. Extent of the depletion for the endogenous chaperones was
assessed by immunoblot analysis of the reticulocyte lysates (0.5 µl each, ~50 µg protein). Protein molecular mass markers (rainbow-colored
markers; Amersham) are myosin (200K), phosphorylase b (97K), serum albumin (69K), ovalbumin (46K), and carbonic anhydrase (30K).
[View Larger Versions of these Images (29 + 66 + 52K GIF file)]
). The molar ratio of hsc70/dj2/dj1 in HepG2
human cell line is 30:4:5 µM.
that anti-hsp40 (dj1) antibody coprecipitated hsc70. When
the lysate was treated with the anti-dj2-coupled resin,
more than 85% of dj2 was removed (lane 3). Similarly,
when the lysate was treated with the anti-dj1-coupled
resin, more than 85% of dj1 was removed (lane 4). However, little decrease of hsc70 was observed in the dj1-
depleted lysate. Intracellular concentration of hsc70 is
much higher than that of dj1 (Fig. 1 b). Thus, a portion of
hsc70 is indeed tightly associated with dj1, while the remaining portion of hsc70 is not. Depletion of either one of
these two DnaJ homologues caused little reduction of the
remaining two chaperones.
, 1996
).
The reason for the decrease is unknown. Import of pOTC
synthesized in the hsc70-depleted lysate was about one-third that of the precursor synthesized in the untreated lysate (Fig. 2 a). When purified mouse hsc70 (130 µg/ml,
1.8 µM) was readded to the hsc70-depleted lysate before
translation, the decreased pOTC import was almost completely recovered. Addition of hsc70 after translation and
before import assay was not effective (Terada et al., 1995
).
Immunoblot analysis ruled out dj1 contamination in our
hsc70 preparation (data not shown). These findings indicate a role for hsc70, but not dj1, in pOTC import into mitochondria.
Fig. 2.
Effect of depletion
and readdition of chaperones
on import of pOTC into rat
liver mitochondria. (a) Rat
pOTC synthesized in the untreated rabbit reticulocyte lysate (Untreated, 15 KBq) or in the hsc70-depleted lysate
without readdition (Depleted, 2.0 KBq) or with readdition (Depleted + Hsc70,
2.0 KBq) of 1.8 µM mouse
hsc70 before translation, was
subjected to import assay, as
described in Materials and
Methods. (b) Rat pOTC synthesized in the mock-depleted
lysate (Mock-Depleted, 23 KBq) or in the dj2-depleted
lysate without readdition
(Depleted, 20 KBq) or with readdition (Depleted + His-dj2, 16 KBq) of 0.4 µM His-dj2 before translation was subjected to import assay. (c) Rat pOTC synthesized in the mock-depleted lysate (Mock-Depleted, 23 KBq) or in the dj1-depleted lysate without readdition (Depleted, 14 KBq) or with readdition (Depleted + His-dj1, 16 KBq) of 0.5 µM His-dj1 before translation was subjected to import assay. Portions of the fluorograms are
shown in the upper part of each panel. p, precursor form of OTC; m, mature form of OTC; 30%, 30% of the input pOTC. The results were quantitated by imaging plate analysis using FUJIX BAS2000 analyzer and are shown in the lower part.
[View Larger Versions of these Images (31 + 32 + 33K GIF file)]
). Since posttranslational addition of the antibody had no effect on mitochondrial
import, dj2 is apparently required during pOTC synthesis
but not during import, the results being similar to those for
hsc70 (Terada et al., 1995
). Requirement of dj2 was further confirmed by the addition of baculovirus-expressed
His-dj2 to the dj2-depleted lysate. The pOTC import was
almost completely recovered when His-dj2 was added before translation. The restoration of pOTC import depended on the amount of added His-dj2, and a nearly complete restoration was achieved with 0.4 µM (data not shown).
This concentration was about double that in the reticulocyte lysate and was about one-tenth of that estimated for
cultured cells and rat liver (see above). Thus, dj2, not dj1,
is the partner DnaJ homologue of hsc70 facilitating mitochondrial import of pOTC.
). We speculated that dj2 participates in stabilization of precursor proteins targeted to the mitochondria in an import-competent
state, whereas dj1 facilitates productive folding of proteins
in the cytosol. Thus, these two DnaJ homologues have distinct roles depending on the final destination of their protein substrates. To test this hypothesis, we investigated
chaperones required for productive folding of luciferase,
using our depletion-readdition system.
). When the reaction was conducted in the hsc70-depleted lysate, refolding was markedly decreased. The residual low refolding
activity in the hsc70-depleted lysate may be attributed to
remaining chaperones other than hsc70 and dj1. When purified hsc70 (1.8 µM) was supplemented to the hsc70-
depleted lysate before the refolding assay, the refolding
activity recovered almost completely. It is to be noted that
this recovery was independent of dj1 that was also removed from the lysate by the hsc70-depletion procedure
(see above).
Fig. 3.
Effect of depletion
and readdition of chaperones
on refolding of chemically denatured luciferase. (a) Chemically denatured luciferase was
renatured in the untreated rabbit reticulocyte lysate (Untreated) or in the hsc70-depleted
lysate without readdition (Depleted) or with readdition (Depleted + Hsc70) of 1.8 µM
mouse hsc70. For comparison,
luciferase was renatured in the
absence of the lysate (Buffer).
(b) Denatured luciferase was
renatured in the mock-depleted
lysate (Mock-Depleted) or in
the dj2-depleted lysate without
readdition (Depleted) or with
readdition (Depleted + His-dj2)
of 0.2 µM His-dj2. Luciferase
was also renatured in the absence of the lysate (Buffer). (c)
Denatured luciferase was renatured in the mock-depleted lysate (Mock-Depleted) or in the dj1-depleted lysate without readdition (Depleted) or with readdition (Depleted + His-dj1) of 0.4 µM His-dj1. Luciferase was also renatured in the absence of the lysate (Buffer). (d) Refolding of luciferase was
conducted for 60 min at 25°C in the dj2-depleted lysate supplemented with decreasing amounts of His-dj2 (0.4, 0.2, 0.1, and 0.03 µM) or
E. coli DnaJ (0.4, 0.2, 0.1, and 0.03 µM). (e) Refolding of luciferase was conducted for 90 min in the hsc70-depleted lysate supplemented
with mouse hsc70 (1.8 µM), His-dj2 (0.4 µM), His-dj1 (0.5 µM), or DnaJ (0.5 µM).
[View Larger Versions of these Images (25 + 27 + 25 + 50 + 32K GIF file)]
). Although the cytosolic hsp70 system of yeast
is complicated (Craig et al., 1995
) and differs from that of
mammals, it may be that the combination of mammalian
hsc70 and bacterial DnaJ will refold luciferase. As shown
in Fig. 3 d, bacterial DnaJ could replace dj2 when added to the dj2-depleted lysate. The amount of DnaJ required
for the refolding of luciferase was similar to that of His-dj2.
At the highest concentration tested, the refolding activity
of DnaJ exceeded that of His-dj2. DnaJ was not effective
in luciferase refolding, when added to the hsc70-depleted
lysate (Fig. 3 e). Thus, the activity of DnaJ depends on the
presence of hsc70.
Fig. 4.
Refolding of luciferase by purified chaperones. (a)
Chemically denatured luciferase was renatured for 90 min at 25°C
in the presence of each chaperone or their combinations. Concentrations of proteins added were: hsc70, 1.8 µM; His-dj2, 0.4 µM; His-dj1, 0.5 µM; DnaJ, 0.5 µM; bovine serum albumin, 1.8 µM. (b) Refolding was performed for indicated periods in the
presence of indicated chaperone(s).
[View Larger Versions of these Images (28 + 24K GIF file)]
reported that hsc70-dj1 prevented thermally denatured luciferase from insoluble aggregates, though no restoration
of the enzyme activity was observed. It seems likely that
the hsc70-dj1 chaperone system maintains thermally denatured luciferase in a refolding-competent state. Interestingly, they also reported the presence of a component(s) in
the reticulocyte lysate that is required for productive folding of luciferase. dj2 may well be a candidate.
Discussion
) with bacterial DnaJ in luciferase folding indicates the universal importance of these limited members of DnaJ family in living cells.
), while
the other reported hsp40 (dj1)-hsc70 only prevented thermally denatured luciferase from insoluble aggregate without recovery of the enzyme activity (Minami et al., 1996
).
In the former report, hdj-1 (dj1) was prepared from E. coli
expressing human dj1, according to procedures for DnaJ
preparation, and the endogenous DnaJ might have been
present in their preparation. In fact, a large amount of the
hdj-1 preparation (1.6 µM) was needed for the refolding of
chemically denatured proteins (Freeman and Morimoto,
1996
; Freeman et al., 1995
). The molar ratio of hdj-1 to
hsc70 used in their study for refolding was 2:1, a ratio
much higher than that in our studies (~1:5, see below) and
that in living cells (Fig. 1 b).
). We also tested effects of dj2-
depletion on mitochondrial import of several precursor
proteins. The requirement of dj2 correlated well with that
of hsc70 (Terada, K., and M. Mori, unpublished observations). The differences in hsc70-dj2 dependency may be due to different tendencies of the precursor proteins to
fold, misfold, or aggregate. A high hsc70 and dj2 dependency of pOTC for mitochondrial import may be reflected
by the high tendency of purified recombinant pOTC to aggregate (Murakami et al., 1990
). We speculate cotranslational interaction of hsc70 and dj2 exists. We reported that
pOTC synthesized in the lysate rapidly looses its import
competence though there are large amounts of hsc70 and
dj2 (Terada et al., 1995
). Neither addition of antibodies against hsc70 and dj2 nor readdition of these chaperones
to the depleted lysate before import reaction affect import
of pOTC synthesized in vitro (Terada et al., 1995
; Kanazawa
et al. 1997
). Thus at least these chaperones are not required at the step of translocation.
). In E. coli, DnaK-DnaJ-GrpE promotes not only folding but also export of proteins in a
SecB-deficient strain (Wild et al., 1992
, 1996
). A common
functional mechanism of hsp70-DnaJ family systems may
be to stabilize unfolded proteins and to protect them from
unproductive folding and aggregation. However, cytosolic
proteins must be folded into their final conformations whereas mitochondrial proteins must be maintained in an
import-competent unfolded conformation (Schatz and Dobberstein, 1996
). We reported that sedimentation coefficients
of import-competent forms of mitochondrial precursor proteins differ from those of their folded mature forms (Terada
et al., 1996
). In the case of 3-oxoacyl-CoA thiolase, the native form in the mitochondrial matrix is a tetramer, whereas the import-competent form apparently behaves as a monomer (Terada et al., 1996
). Thus, mitochondrial precursor
proteins must bypass the cytosolic folding pathway that is
composed of hsc70 and probably dj2 and TriC complex.
This may be achieved by the presence of NH2-terminal
presequences and the presequence-specific factors (PBF
or MSF) that may prevent further folding of the precursor proteins in the cytosol (Mihara and Omura, 1996
). MSF was
reported to depolymerize and unfold aggregated precursor proteins in the presence of ATP and to maintain import competence of precursor proteins (Hachiya et al., 1993
).
Cooperation of these factors with the hsc70-dj2 system in
mitochondrial protein import remains to be elucidated.
; Atencio and Yaffe, 1992
), then it was shown to be a partner
chaperone of Ssa1p (Cyr and Douglas, 1994
; Becker et al.,
1996
). Ydj1p was also shown to be involved in signal transduction pathways mediated by steroid receptors and v-Src
tyrosine kinase (Caplan et al., 1995
; Kimura et al., 1995
;
Dey et al., 1996
; Chang et al., 1997
) and in a ubiquitin- dependent protein degradation pathway (Lee et al., 1996
;
Yaglom et al., 1996
). In mammals, these functions may
possibly be maintained by dj1 and/or dj2.
Received for publication 18 August 1997 and in revised form 6 October 1997.
1. Abbreviations used in this paper: dj1, mammalian counterpart of hsp40/ hdj-1; dj2, mammalian counterpart of HSDJ/hdj-2; His-dj1 and His-dj2, hexahistidine-tagged dj1 or dj2; hsc70, 70K heat shock cognate protein; pOTC, preornithine transcarbamylase.We thank K. Ohtsuka of Aichi Cancer Center Research Institute and Y. Minami of Oita Medical College for the generous gift of the His-hsp40 expression system and N. Imamoto and Y. Yoneda of Osaka University for human recombinant hsc70. We also thank T. Ogura, M. Takiguchi, and other colleagues of Kumamoto University for suggestions and technical advice and M. Ohara for comments on the manuscript.
This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (09276103 and 08457040 to M. Mori and 09780658 to K. Terada).
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