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(Received for publication, March 26, 1996, and in revised form, May 29, 1996)
From the Howard Hughes Medical Institute and Cellular Biochemistry
and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New
York, New York 10021 and § Laboratory of Experimental
Radiology, Aichi Cancer Center Research Institute, Chikusa-ku,
Nagoya 464, Japan
ABSTRACT The effects of the human DnaJ homolog, Hsp40, on
the ATPase and chaperone functions of the constitutively expressed
Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of
ATP by Hsc70, causing a ~7-fold increase in its steady-state ATPase
activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis
and not the release of bound ADP is the rate-limiting step in the
overall ATPase cycle of mammalian Hsc70. The ability to activate the
Hsc70 ATPase is partially preserved in a deletion mutant containing the
J-domain and the G/F region of Hsp40 but not in a deletion mutant that
contains the J-domain alone. As a result of its ATPase stimulating
activity, addition of Hsp40 allows Hsc70 to bind peptide in the
presence of ATP, whereas in the absence of Hsp40, peptide is
efficiently released upon ATP binding to Hsc70. The functional
cooperation of Hsp40 with Hsc70 is essential to ensure the ATP
hydrolysis-dependent binding of aggregation-sensitive denatured
polypeptides, such as thermally denatured firefly luciferase and
chemically denatured rhodanese. Binding of these proteins results in
the formation of ternary complexes of Hsc70, Hsp40, and substrates.
Hsc70 and Hsp40 cooperate with further factors in protein renaturation,
as demonstrated by the finding that luciferase, thermally denatured in
the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit
reticulocyte cytosol. Our results indicate that Hsp40 has a critical
regulatory function in the Hsc70 ATPase cycle that is required for the
efficient loading of peptide substrate onto Hsc70.
INTRODUCTION The folding of many newly synthesized polypeptides in the cell is
assisted by molecular chaperones, a class of proteins which function
mainly in preventing off-pathway folding reactions that lead to
aggregation (1, 2, 3). Two major chaperone families, the 70-kDa heat-shock
proteins (Hsp70s) and the chaperonins, play a central role in these
reactions in the cytosol and within organelles (2, 3). Among other
functions, Hsp70 family members are thought to prevent the misfolding
of translating polypeptides. This is accomplished by their ability to
bind extended peptide segments, preferentially 7-mer or 8-mer peptides
which exhibit a certain enrichment and pattern of hydrophobic amino
acid residues (4, 5, 6, 7). Such sequences are probably exposed during
translation but are buried within the core of the folded protein. The
chaperonins act downstream from the Hsp70s by sequestering partially
folded intermediates within a central cavity and promoting their
folding to the native state (2, 3).
Hsp70 proteins consist of two domains, a highly conserved N-terminal
ATPase domain of ~45 kDa (8, 9) and a C-terminal domain of 25 kDa. An
18-kDa portion of the C-terminal domain adjacent to the ATPase domain
contains the polypeptide binding site (10, 11). The chaperone activity
of the Hsp70 family is controlled by a reaction cycle of ATP binding,
hydrolysis, and nucleotide exchange. The ATP-bound form of Hsp70 binds
and releases peptide rapidly, resulting in a low overall affinity,
whereas the ADP form binds peptide slowly but more stably (12, 13, 14, 15). ATP
binding to the ATPase domain causes a conformational change, which in
turn results in structural alterations in the C-terminal domain leading
to substrate release (12, 16, 17, 18, 19). Since the intrinsic ATPase activity
of Hsp70 proteins is low, the ATP-bound form with low substrate
affinity predominates (12, 13, 14, 15). As a consequence, the chaperone
activity of Hsp70 depends on its functional regulation by cofactors
that catalyze the interconversion between the ATP and ADP states. Among
these factors are the DnaJ and GrpE proteins of Escherichia
coli (15, 20, 21, 22, 23, 24). DnaJ accelerates the rate of ATP-hydrolysis of
the bacterial Hsp70, DnaK, whereas GrpE promotes nucleotide exchange
(15, 21, 22). Acting in concert, DnaJ and GrpE stimulate the ATPase
activity of DnaK by up to 50-fold or more (22). In addition, DnaJ has
the ability to bind unfolded polypeptide on its own (20, 21, 23, 24)
and may target a substrate protein to DnaK in its ATP-bound state. This
leads to the formation of a ternary complex of DnaJ, DnaK (in the ADP
state), and polypeptide (21, 23). The complex dissociates upon
GrpE-catalyzed ADP release and subsequent ATP rebinding. At this point,
substrate protein is set free and has the option to fold, to be
transferred to another chaperone system, or to bind back to DnaJ and
DnaK (3).
The functional regulation of the eukaryotic Hsp70 system is less well
understood. Homologs of DnaJ exist in all compartments which contain
Hsp70 (25, 26, 27, 28, 29, 30), and several typical family members have been identified
in mammalian cells (25, 26, 30). However, a eukaryotic GrpE homolog has
so far been found only in mitochondria (31). It has therefore been
speculated that the Hsp70 proteins in the eukaryotic cytosol may be
regulated differently from DnaK. Here, we have characterized the role
of the human DnaJ homolog, Hsp40 (Hdj1), in the reaction cycle of
Hsc70, the constitutively expressed Hsp70 in the mammalian cytosol. Our
results show that Hsp40 stimulates the Hsc70 ATPase by increasing the
rate of ATP hydrolysis. Hsp40 is required to allow the efficient,
ATP-dependent binding of polypeptide substrate to Hsc70.
Thus, the chaperone function of Hsc70 in preventing protein aggregation
and in mediating the renaturation of unfolded polypeptide is critically
dependent on the cooperation with Hsp40.
MATERIALS AND METHODS Plasmid Constructions
The DNA fragment used to introduce an
NdeI site at the initiation methionine of Hsp40 was
constructed via a polymerase chain reaction
(PCR)1 using an Nde-primer
[5
The DNA fragment used to introduce a
BamHI site just prior to the initiation methionine in
pBSII-Hsp40 was constructed via PCR by using two primers, a
Bam primer (5 The DNA fragment used to introduce a
SalI site at methionine-125 of Hsp40 was constructed via PCR
by using a Bam primer and a Sal primer
(5 Protein Purification
To purify nontagged Hsp40, the plasmid pET/Hsp40 was transformed
into BL21(DE3) cells grown at 37 °C. After a 2-h incubation with 0.4 mM isopropyl-1-thio- To purify the 6His-tagged proteins, the plasmids pQE-9/Hsp40 and
pQE-9/40(J+G/F) were transformed into SG13009 cells and grown at
37 °C. After a 1-h induction with 0.1 mM IPTG, cells
were lysed as above in buffer B (50 mM HEPES-KOH, pH 7.5, 300 mM NaCl) containing 1 mM
phenylmethylsulfonyl fluoride. The cleared lysate was loaded onto a
Ni2+-NTA agarose column (QIAGEN Inc.) equilibrated with
buffer B. The column was washed with buffer B, and proteins were eluted
with a linear gradient of 0-500 mM imidazole in buffer B. Peak fractions of His-Hsp40 were collected and further purified by a
combination of hydroxyapatite HTP and DEAE-Sephacel as described above.
Purified Hsp40, His-Hsp40, and His-Hsp40(J+G/F) were concentrated by
ultrafiltration and dialyzed against buffer C (20 mM sodium
phosphate, pH 7.5, 100 mM NaCl, 0.1 mM EDTA).
The purified Hsp40 J-domain (residues 1-77; Hsp40(J) was provided by
D. McColl (52).
Hsc70 was purified from bovine brain according to the method described
by Schlossman et al. (33). DnaK and DnaJ were purified as
described elsewhere (21, 24). Protein concentrations were determined
using the Bio-Rad protein assay.
ATPase Assay
Hsc70 and DnaK were incubated for 30 min at 30 °C in 50-µl
reaction mixtures containing 10 mM MOPS-KOH, pH 7.2, 50 mM KCl, 10 mM MgCl2, and 50 µM [
Substrate Binding Assay
Peptide C from the vesicular stomatitis virus glycoprotein
(KLIGVLSSLFRPK) was 14C-labeled by reductive methylation.
Peptide C (0.9 mg) in 120 µl of 170 mM potassium
phosphate, pH 8.0, was incubated with 6.3 µl of
[14C]formaldehyde (58 mCi/mmol, DuPont NEN) and 20 µl
of 0.2 M NaBH3CN for 3 h at room
temperature. The reaction mixture was subjected to two rounds of gel
filtration on a 1-ml Sephadex G-10 spin column to reisolate the labeled
peptide C. The specific activity of the labeled peptide was 42 cpm/pmol. Hsc70 (5.1 µM) was incubated with
14C-peptide C (36 µM) in buffer D (10 mM MOPS-KOH, pH 7.2, 150 mM KCl, 3 mM MgCl2) in the presence of the indicated
combinations of Hsp40 (10 µM) and nucleotide (30 µM) for 30 min at 37 °C. Hsc70-peptide C complexes
were separated from free peptide by gel filtration on 1-ml Sephadex
G-50 spin columns. Amounts of Hsc70 were quantified by densitometry of
Coomassie-stained SDS-polyacrylamide gels (PAGE). Hsc70-bound peptide C
was quantified by liquid scintillation counting.
Aggregation Assay by Centrifugation
Purified firefly luciferase (0.1 µM;
Sigma) was incubated in buffer E (10 mM
MOPS-KOH, pH 7.2, 50 mM KCl, 3 mM
MgCl2, 2 mM dithiothreitol) containing the
indicated combinations of chaperones (Hsc70, 4.7 µM;
Hsp40, 3.2 µM) and ATP (2 mM) for 5 min at
room temperature, followed by a 10-min incubation at 42 °C. Reaction
mixtures were centrifuged at 16,000 × g for 5 min at
4 °C. Supernatants, precipitated by trichloroacetic acid, and pellet
fractions were analyzed by SDS-PAGE, and the amounts of luciferase were
quantified by densitometry.
Light Scattering Assay
Rhodanese from bovine liver (Sigma) was
denatured at 46 µM in a buffer containing 6 M
guanidinium-HCl, 30 mM MOPS-KOH, pH 7.2, and 2 mM dithiothreitol by incubation for 30 min at room
temperature. Denatured rhodanese was diluted 100-fold (0.46 µM final concentration) into buffer E containing the
indicated combinations of chaperones (Hsc70, 2.8 µM;
Hsp40, 1.8 µM) and nucleotide (2 mM ATP or
AMP-PNP). Aggregation was monitored by measuring the turbidity of the
solution at 320 nm for 10 min at room temperature.
Complex Formation between Hsc70, Hsp40, and Luciferase
Luciferase (1.1 µM) was incubated in buffer D
containing 2 mM dithiothreitol and the indicated
combinations of chaperones (Hsc70, 4.7 µM; His-Hsp40, 3.5 µM) and nucleotides (2 mM ATP, ADP, AMP-PNP,
or ATP Refolding Assay
Luciferase (0.1 µM) was incubated in the presence
or absence of 2 mM ATP in buffer E containing the indicated
chaperone components for 5 min at room temperature followed by thermal
inactivation for 10 min at 42 °C. Refolding was initiated by adding
untreated rabbit reticulocyte lysate (Green Hectares) to 2.5 or 5% and
2.5 mM ATP, and luciferase activities were measured after
incubation at 30 °C for various times using the Promega luciferase
assay system and a Bio-Orbit luminometer (35).
RESULTS Two DnaJ homologs have been identified in the mammalian cytosol,
Hsp40 (Hdj1) and Hdj2 (Hsdj) (25, 26). While Hdj2 contains all three
conserved regions found in E. coli DnaJ, including the
N-terminal ``J-domain,'' the glycine-phenylalanine (G/F)-rich region
and the cysteine-rich zinc finger domain (27, 28, 29, 36, 37, 38, 39), Hsp40 lacks
the zinc finger domain. To analyze the role of Hsp40 in the Hsc70
ATPase cycle, we purified Hsp40 upon expression in E. coli
with and without an N-terminal 6His-tag (Fig. 1). As
shown below, both proteins behaved identically in all functional assays
performed in this study. To determine the minimal region of Hsp40
necessary for the stimulation of the Hsc70 ATPase, two deletion mutants
of Hsp40 were constructed, corresponding to the J-domain plus the G/F
region and the J-domain alone (Fig. 1).
Addition of
Hsp40 to bovine Hsc70 caused a significant stimulation of the Hsc70
ATPase in a dose-dependent manner (Fig.
2A). The maximum stimulation by Hsp40 was
approximately 7-fold with His-Hsp40 being equally effective (Fig.
2B, lanes 2, 8, and 9).
DnaJ of E. coli increased the ATPase activity of bovine
Hsc70 at lower concentrations than Hsp40 (Fig. 2A), although
the maximum stimulation reached with both proteins was similar (Fig.
2B, lanes 2 and 3). Interestingly, the
inverse reaction was not observed; Hsp40 hardly stimulated the ATPase
activity of DnaK (Fig. 2B, lane 5).
In agreement with previous observations, the steady-state ATPase
activity of DnaK was stimulated by DnaJ only 3-fold (Fig.
2B, lane 6), as compared to the 7-fold
stimulation of the Hsc70 ATPase by Hsp40. Since full activation of the
DnaK ATPase requires the cooperation of DnaJ and the nucleotide
exchange factor GrpE (15, 21, 22), it was of interest whether Hsp40
acts on Hsc70 by accelerating the step of ATP hydrolysis or nucleotide
exchange, or both. Hsc70 was incubated with [ The strong stimulation of the Hsc70 ATPase by Hsp40 alone suggested
that the dissociation of ADP may not be the rate-limiting step in the
overall ATPase cycle. This was confirmed by the observation that the
ADP bound to Hsc70 in the presence of Hsp40 was exchanged for ATP about
three times more rapidly than ADP that was bound to DnaK in the
presence of DnaJ. The half-time for the dissociation of ADP from Hsc70
was less than 30 s as compared to approximately 90 s in the
case of DnaK (Fig. 2D). This explains why Hsp40 alone is
able to stimulate the ATPase of Hsc70 without the aid of a GrpE-like
factor. Indeed, in the eukaryotic system the dissociation of ADP from
Hsc70 is even subject to negative regulation. A novel protein termed
Hip (Hsc70 interacting protein) has recently been described that binds
the ATPase domain of Hsc70, thereby slowing the dissociation of ADP
from Hsc70 (34). Taken together, the rate of nucleotide exchange by
Hsc70 appears to be sufficiently fast so that a nucleotide-releasing
factor, such as GrpE, may be dispensable.
The interaction between DnaJ proteins and their respective Hsp70
partners is thought to be mediated mainly by the ~70-amino acid
residue J-domain of DnaJ proteins. However, as shown for E. coli DnaJ, the J-domain alone is necessary but not sufficient to
stimulate the ATPase of DnaK (38, 39). This function requires the
G/F-rich segment in addition to the J-domain. Similar observations were
made here for the interaction between Hsp40 and Hsc70. Whereas the
J-domain of Hsp40 was inefficient in stimulating the hydrolysis of ATP
by Hsc70, a mutant protein containing the J-domain plus the G/F-domain
caused a significant activation of the ATPase equivalent to about 50%
of that seen with full-length Hsp40 (Fig. 3).
Hsc70 is
known to bind extended peptide segments exposed by nascent polypeptides
(35, 40, 41). Peptide C of vesicular stomatitis virus glycoprotein is
bound by Hsc70 with high affinity (42) and was used as a model for a
nascent-chain substrate. The interaction with peptide C is dependent on
the nucleotide bound state of Hsc70 (Fig. 4). Whereas
ATP-bound Hsc70 has a low affinity for the peptide (Fig. 4, lane
2), the ADP-bound (or nucleotide-free) state binds strongly (Fig.
4, lanes 1 and 3). Significantly, efficient
binding of peptide C to Hsc70 was detected in the presence of ATP upon
simultaneous addition of Hsp40. Under the experimental conditions,
~40% of the binding capacity of Hsc70 was reached (Fig. 4,
lane 5). Hsp40 itself showed no measurable affinity for
peptide C in the presence or absence of nucleotide and it seems
unlikely that it acquires such an affinity in the presence of Hsc70. It
appears more likely that the stabilization of the Hsc70-peptide complex
is the result of the stimulation of the Hsc70 ATPase by Hsp40, which
generates the ADP-bound state of Hsc70 with high peptide affinity.
The
effect of Hsp40 on the binding of denatured polypeptides by Hsc70 was
analyzed with thermally unfolded firefly luciferase and chemically
denatured rhodanese as the substrates. In the absence of Hsc70 and
Hsp40, more than 95% of luciferase aggregated during a 10-min
incubation at 42 °C, whether ATP was present or not (Fig.
5A, lanes 7 and 8).
However, when both Hsc70 and Hsp40 were present during thermal
inactivation, 70% of the total luciferase protein remained soluble.
Significantly, this chaperone effect was ATP-dependent
(Fig. 5A, lanes 1 and 2). Either Hsc70
or Hsp40 alone moderately increased the concentration of soluble
luciferase and this effect was enhanced by the addition of ATP (Fig.
5A, lanes 3-6). However, only the luciferase
that had been stabilized by both Hsc70 and Hsp40 was competent for
subsequent reactivation (see below).
The cooperative effect of Hsc70 and Hsp40 in stabilizing unfolded
polypeptide was more clearly observed with rhodanese, a protein that
aggregates rapidly upon dilution from guanidinium HCl (43, 44). The
combination of Hsc70 and Hsp40 effectively prevented the aggregation of
rhodanese, as measured by light scattering (Fig. 5B). This
effect was apparently dependent on ATP hydrolysis, because the
nonhydrolyzable analog AMP-PNP could not substitute for ATP.
Significantly, Hsc70 alone prevented the aggregation of rhodanese only
slightly, whereas Hsp40 alone was without a stabilizing effect. We
conclude that Hsp40 and ATP are required for the efficient binding of
unfolded polypeptide by Hsc70, in particular with substrates that
undergo rapid aggregation.
The requirement of Hsp40 for efficient
substrate binding by Hsc70 suggested the possibility that Hsp40 may
induce the formation of ternary complexes containing Hsc70, Hsp40, and
unfolded substrate protein. Purified 6His-tagged Hsp40 served to test
this hypothesis. Upon incubation at 42 °C, His-Hsp40, Hsc70, and
luciferase formed a complex that could be isolated on
Ni2+-NTA-agarose (Fig. 6A,
lanes 1 and 2). Complex formation was dependent
on ATP. His-Hsp40 alone did not stably bind luciferase (Fig.
6A, lane 4) and in the absence of His-Hsp40
neither Hsc70 (data not shown) nor luciferase (Fig. 6A,
lane 3) was retained on Ni2+-NTA-agarose.
Efficient complex formation apparently required the hydrolysis of ATP,
although the complex formed with approximately 40% efficiency in the
presence of AMP-PNP. ADP and the nonhydrolyzable analog ATP
Although
purified Hsc70 and Hsp40 cooperated in an ATP-dependent
manner in preventing the formation of insoluble aggregates of
luciferase at 42 °C, we did not observe a regain of luciferase
activity upon temperature downshift to 30 °C (Fig.
7A, closed triangles). Efficient
reactivation with a half-time of ~10 min occurred, however, when
rabbit reticulocyte cytosol was added after the thermal inactivation
step. As a prerequisite for successful reactivation, Hsc70, Hsp40, and
ATP had to be present during thermal inactivation (Fig. 7A,
closed circles). Bovine serum albumin could not substitute
for the chaperones during inactivation (Fig. 7B, lane
6). Neither was efficient reactivation observed when the omitted
component(s) was added back after thermal inactivation (Fig.
7C). Purified Hip did not show a significant effect on the
interaction between Hsc70/Hsp40 and thermally denatured luciferase
(data not shown). We conclude that Hsc70, Hsp40, and ATP are necessary
during thermal inactivation to maintain luciferase in a soluble,
refolding-competent state. A preliminary characterization of the
additional lysate factor(s) required for renaturation indicated that it
does not have the properties of a GrpE-like component but rather
represents another chaperone system that cooperates with Hsc70/Hsp40,
perhaps in dissociating small aggregates of luciferase (see
``Discussion'').
Reactivation of thermally inactivated
luciferase. A, luciferase (0.1 µM) was
incubated for 10 min at 42 °C in the presence of Hsc70 (4.7 µM) and Hsp40 (3.2 µM) with or without 2 mM ATP and then incubated at 30 °C with or without 5%
reticulocyte lysate in the presence of freshly added 2.5 mM
ATP. Luciferase activities were measured before and after the
incubation at 42 °C and are expressed as percent of the activity
before thermal inactivation. B, luciferase (0.1 µM) was inactivated at 42 °C as above in the presence
of the components indicated followed by the addition of 2.5%
reticulocyte lysate and 2 mM ATP. Lane 1, ATP;
lanes 2 and 3, 4.7 µM Hsc70 and 3.2 µM Hsp40 with and without ATP, respectively; lane
4, 7.6 µM Hsc70 and ATP; lane 5, 7.9 µM Hsp40 and ATP; lane 6, 7.4 µM
bovine serum albumin and ATP. Luciferase activities were measured after
60-min incubation at 30 °C. C, luciferase (0.1 µM) was inactivated at 42 °C in the presence of the
indicated combinations of Hsc70 (4.7 µM), Hsp40 (3.2 µM) and ATP (2 mM) (during thermal
inactivation) followed by supplementation of the omitted component(s) (after thermal inactivation).
Luciferase activities were measured 60 min after addition of 2.5%
reticulocyte lysate and 2.5 mM ATP.
DISCUSSION Hsc70 and Hsp40 have been implicated in protecting translating
polypeptide chains against aggregation (35), but the mechanism of Hsp40
action in this reaction has been unclear so far. Our analysis with
purified chaperone proteins showed that Hsp40 significantly enhances
the ATPase activity of Hsc70, thereby allowing rapid and efficient
binding of Hsc70 to unfolded substrate proteins. We propose that this
function of Hsp40, and perhaps other DnaJ homologs, is required for the
loading of Hsc70 onto a nascent polypeptide as it emerges from the
ribosome or for the efficient binding of Hsc70 to a polypeptide that
has been denatured under heat stress.
Our analysis revealed interesting differences in the ATPase cycles of
the eukaryotic and prokaryotic Hsp70 systems. Since the spontaneous
release of ADP from Hsc70 is more rapid than from DnaK (Fig.
2D), the stimulation of ATP-hydrolysis by Hsp40 is alone
sufficient to produce a significant acceleration of the steady-state
ATPase of Hsc70. In contrast, the rate-limiting step in the DnaK ATPase
cycle is the dissociation of ADP (15, 21, 22), explaining the
dependence of the prokaryotic Hsp70 system on an additional nucleotide
exchange factor, GrpE. Despite considerable effort, such a factor has
not been identified in the eukaryotic cytosol. In fact, the
dissociation of ADP from eukaryotic Hsp70 is even subject to negative
regulation. The recently identified cochaperone of Hsc70, Hip,
stabilizes the ADP-state of Hsc70, thereby probably slowing the
dissociation of Hsc70-substrate complexes (34). These observations
suggest that Hsp40 may be sufficient to accelerate the Hsc70 ATPase for
efficient binding of substrate proteins. Other eukaryotic DnaJ
homologs, including Saccharomyces cerevisiae Ydj1p (45, 46, 47)
and human Hsj1 (48), have also been reported to stimulate the ATPase
activity of Ssa1p and Hsc70, respectively, without the aid of a
GrpE-like factor. The nearly complete S. cerevisiae genome
contains no sequence homolog of GrpE except for mitochondrial GrpE.
Likewise, a recently published list of human cDNA sequences
contains only one sequence coding for a putative GrpE homolog, which
probably resides in mitochondria (49). In light of the apparent
dispensability of a GrpE-like protein in the cytosol, it may be
relevant that the cytosolic Hsp70s of eukaryotes contain the conserved
C-terminal tetrapeptide EEVD, which has been implicated in the
intramolecular regulation of Hsc70 function and in its interactions
with Hsp40 (18).
The N-terminal J-domain is the signature domain of all members of the
DnaJ family and is thought to be largely responsible for the
interaction between DnaJ homologs and their Hsp70 partners (26, 27, 28, 29).
The three-dimensional structure of the J-domain consists of a
scaffolding of four The stimulation of the Hsc70 ATPase by Hsp40 resulted in a significant
enhancement of the substrate binding activity of Hsc70 in the presence
of ATP. Rapid peptide binding is known to occur in the ATP-bound state
of Hsp70, but the complex is only stable when the ADP-state of Hsp70 is
generated (this study) (12, 15, 21, 23). Whereas free peptide could
also form a complex with the ADP-bound or nucleotide-free forms of
Hsc70 directly, the full requirement of ATP and Hsp40 for substrate
binding became apparent with completely unfolded polypeptide substrates
that undergo rapid aggregation. Rapid binding of Hsp70 may be
particularly relevant during the exposure of cells to elevated
temperatures, as shown here for firefly luciferase. The ADP-bound and
nucleotide-free states of Hsp70 were not capable of preventing the
aggregation of the unfolded protein, apparently because their on-rate
for binding is too slow. We conclude that the Hsp40-catalyzed
conversion of Hsc70-bound ATP to ADP is absolutely critical for the
binding of aggregation-prone substrates to Hsc70 and thus for the
physiological function of the Hsc70-system, both during translation and
under conditions of cellular stress.
E. coli DnaJ (21, 23, 24, 39) and the DnaJ homolog Ydj1p of
S. cerevisiae (51) have the capacity to bind unfolded
proteins and are thus classified as molecular chaperones. It has been
proposed that during the DnaK/DnaJ/GrpE reaction cycle DnaJ binds first
to the unfolded polypeptide substrate and presents it to ATP-bound DnaK
(15, 21). Interestingly, the substrate binding specificity of DnaJ
differs from that of DnaK in that DnaJ is apparently unable to bind
extended peptide segments but rather interacts with protein folding
intermediates that expose hydrophobic surfaces (23). We were unable to
convincingly demonstrate a similar capability of Hsp40 to bind purified
unfolded polypeptide (Figs. 5, 6, 7). On the other hand, nascent
polypeptide chains have been coimmunoprecipitated with antibodies
against Hsp40 from a reticulocyte lysate translation, even under
conditions where Hsc70 was relatively depleted (35). It is thus likely
that Hsp40 has at least a weak affinity for unfolded polypeptide that
may explain the observed formation of ternary complexes between
unfolded luciferase, Hsp40 and Hsc70 (Fig. 6). In support of this
notion, the deletion mutant of Hsp40 containing the J-domain and the
G/F region retained a partial activity to stimulate the Hsc70 ATPase
but has lost the ability to form a ternary complex with substrate
protein and Hsc70.2 Thus, the C-terminal
region of Hsp40 is likely responsible for this activity. This part of
the molecule is distinct from the corresponding region of DnaJ in that
it lacks the ~80 residue cysteine-rich zinc finger domain that has
recently been shown to mediate binding of unfolded polypeptides (39).
This may be responsible for the strongly reduced activity of Hsp40 to
stabilize unfolded polypeptide in the absence of Hsc70. It is of
interest in this respect that Hdj2, the other DnaJ homolog in the
mammalian cytosol (26), contains a cysteine-rich central domain. The
functional cooperation between Hsc70 and Hdj2 has not yet been analyzed
but we speculate that Hdj2 may have chaperone properties similar to
those of E. coli DnaJ.
We have recently reported that Hsc70 and Hsp40 can cooperate with Hip
in refolding denaturant-unfolded luciferase in an
ATP-dependent manner (34). Increasing the concentrations of
Hsc70 and Hsp40 relative to luciferase can compensate for the
requirement of Hip.2 Hip has been shown to stabilize the
ADP-bound form of Hsc70, induced by Hsp40, and thus maintains the high
affinity state of Hsc70 for substrate proteins. The Hsc70-substrate
complex is otherwise labile because of the spontaneous dissociation of
ADP (34). This would explain why Hip reduces the amounts of Hsc70 and
Hsp40 required for efficient refolding of chemically denatured
luciferase.
Although Hsc70 and Hsp40 protected luciferase from aggregation at
elevated temperature by forming a ternary complex, the bound luciferase
was unable to refold upon temperature downshift (Fig. 7). Only upon
addition of reticulocyte cytosol was the efficient renaturation of this
protein observed. Preliminary experiments to purify this factor showed
that the refolding-stimulating activity fractionates at ~600 kDa on a
size-exclusion column, distinct from the large chaperonin TRiC. It may
represent a new chaperone activity or a combination of known chaperone
components that remains to be defined.
FOOTNOTES Acknowledgments We are grateful to Morten Søgaard for
supplying plasmid pMS/MycHis, purified Hsc70, and peptide C. We thank
Damian McColl for critically reading the manuscript.
REFERENCES
Volume 271, Number 32,
Issue of August 9, 1996
pp. 19617-19624
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
-CCGCAGGAGGGGCATATGGGTAAAGAC-3] and an Nco-primer
[5-GAGGGTCTCCATGGAATGTGTAGCTG-3]. The latter included an
NcoI site corresponding to nucleotide 322 of the human Hsp40
cDNA clone, pBSII-Hsp40 (25), which was used as the template. The
Hsp40-coding region of pBSII-Hsp40 was digested with BamHI
and SacI and inserted into the complementary sites in the
modified plasmid pET-3a (Novagen, Inc.). The PCR-amplified DNA was
digested with NdeI and NcoI, and replaced the
NdeI-NcoI region of the above plasmid to create
the plasmid pET/Hsp40, expressing nontagged Hsp40 (Fig. 1,
Hsp40).
Fig. 1.
Schematic representation of wild type Hsp40
and the deletion mutants of Hsp40 proteins used in this study. The
J-domain is a ~70-amino acid region which is highly conserved among
all DnaJ homologs. G/F is a domain rich in glycine and
phenylalanine.
-CCGCAGGAGGGATCCATGGGTAAAGAC-3) and an
Nco-primer. The PCR-amplified DNA was digested with
BamHI and NcoI and replaced the
BamHI-NcoI fragment of pBSII-Hsp40. The entire
coding region of Hsp40 was excised with BamHI and
PstI and inserted into the complementary sites in pQE-9
(QIAGEN Inc.) to create the plasmid pQE-9/Hsp40, expressing
histidine-tagged Hsp40 (Fig. 1, His-Hsp40).
-GTCATCAATGTCGACGCCTTCCTC-3) and pBSII-Hsp40 as a template. The
PCR-amplified DNA was digested with BamHI and
SalI and inserted into the complementary sites in pQE-9 to
create the plasmid pQE-9/40(J+G/F), expressing the truncated
protein His-Hsp40(J+G/F) (Fig. 1). All DNA fragments amplified by PCR
were sequenced. Standard molecular biology techniques were followed as
described previously (32) or recommended by the manufacturers.
-D-galactoside (IPTG),
cells were lysed in a French pressure cell (SLM Instruments, Inc.) in
buffer A (20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 1 mM EDTA) containing 1 mM phenylmethylsulfonyl
fluoride. The cleared lysate was mixed with DEAE-Sephacel (Pharmacia
Biotech Inc.) on ice for 1 h. The unbound material was collected,
and the resin was washed with buffer A. The flow-through and first wash
were combined and loaded onto a hydroxyapatite HTP column (Bio-Rad)
equilibrated with 100 mM potassium phosphate, pH 7.6. The
column was washed with the same buffer, and Hsp40 was eluted with a
linear gradient of 100-300 mM potassium phosphate, pH 7.6. Peak fractions were rechromatographed on an HTP column after passing
them through a DEAE-Sephacel column.
-32P]ATP (2.5 µCi; 3000 Ci/mmol,
Amersham Corp. or DuPont NEN). 2-µl samples were assayed for ADP
formation by thin layer chromatography on Polygram CEL 300 polyethyleneimine cellulose plates (Macherey-Nagel) (16, 21). The
relevant spots were excised and their radioactivity determined by
liquid scintillation counting to determine rates of ATP hydrolysis. To
analyze the effects of Hsp40 on the nucleotide-bound state of Hsc70,
Hsc70-32P-nucleotide complexes were formed as follows. 17.5 µM Hsc70 was incubated in a 100-µl reaction mixture
containing 100 µM [-32P]ATP (10 µCi)
for 10 min at room temperature and then loaded onto a 1-ml Sephadex
G-50 spin column (34). Hsc70-32P-nucleotide complexes were
recovered by centrifugation for 2 min and divided into two portions
(containing about 3 µM Hsc70), to which Hsp40 (5 µM) and buffer C were added, respectively. After a 1-min
incubation at room temperature, Hsc70-bound nucleotide was again
separated from free nucleotide by spin-column chromatography. To
analyze the dissociation of ADP bound to Hsc70 or DnaK, Hsc70- or
DnaK-32P-nucleotide complexes were formed as described
above. Within a 1-min incubation of the respective complex with Hsp40
(4.6 µM) or DnaJ (4.8 µM) at room
temperature, all the nucleotide was converted to ADP (data not shown;
cf. Fig. 2C). The obtained Hsc70- or
DnaK-[-32P]ADP complexes were incubated with 0.1 mM ATP at room temperature for the times indicated,
immediately followed by spin-column chromatography to recover the
[-32P]ADP bound to Hsc70 or DnaK.
Fig. 2.
Stimulation of the Hsc70 ATPase by Hsp40.
A, Hsc70 (1.4 µM) and His-Hsp40 or DnaJ were
incubated at varying concentrations with [-32P]ATP (50 µM) for 30 min at 30 °C. B, the ATPase
activity of 1.4 µM Hsc70 (lane 1) or 0.5 µM DnaK (lane 4) was stimulated by saturating
concentrations of His-Hsp40 (lane 2, 3.9 µM;
lane 5, 1 µM) and DnaJ (lane 3, 0.5 µM; lane 6, 1 µM). Results are
presented as fold stimulation relative to the basal ATPase activities
of Hsc70 (0.20 nmol/min/mg) or DnaK (2.5 nmol/min/mg), respectively.
Saturating concentrations (2 µM) of both 6His-tagged
(lane 8) and nontagged Hsp40 (lane 9) stimulated
the Hsc70 ATPase (lane 7) to the same extent. C,
Hsp40 stimulated the conversion of Hsc70-bound ATP to ADP, but not the
release of Hsc70-bound ADP. The Hsc70-32P-nucleotide
complex (lane 1) was incubated without (lane 2)
or with Hsp40 (lane 3) for 1 min at room temperature and
passed through a Sephadex G-50 spin column to separate free from
Hsc70-bound nucleotide (lanes 4 and 5, without
and with Hsp40, respectively). D, dissociation of
[-32P]ADP from Hsc70/Hsp40 or DnaK/DnaJ observed as a
function of time.
S) for 5 min at room temperature followed by a 10-min
incubation at 42 °C. After removal of large aggregates by
centrifugation, supernatants were mixed with 20 µl of
Ni2+-NTA-agarose beads on ice for 30 min. Beads were washed
sequentially with 500 µl of buffer B containing 3 mM
MgCl2 and buffer B containing 3 mM
MgCl2 and 50 mM imidazole. When present during
complex formation, the respective nucleotides were also added to the
wash buffer. Proteins retained on the beads were finally eluted with
500 µl of buffer B containing 150 mM imidazole,
trichloroacetic acid-precipitated, and analyzed by SDS-PAGE. The amount
of luciferase in each reaction was quantified by densitometry.
-32P]ATP
for 10 min and then removed from free nucleotide by rapid gel
filtration. A mixture of [-32P]ATP- and
[-32P]ADP-bound Hsc70 was isolated (Fig.
2C). When this reaction was incubated with Hsp40 for 1 min,
almost all of the Hsc70-bound ATP was converted to ADP (Fig.
2C, lane 3). Reisolation of the Hsc70 revealed
that the ADP generated was still bound to the same extent as in the
control reaction that had not been incubated with Hsp40 (Fig.
2C, compare lanes 2 and 3 to
lanes 4 and 5, respectively). We conclude that
Hsp40 does not significantly increase the rate of nucleotide
dissociation from Hsc70 but rather catalyzes the hydrolysis of
Hsc70-bound ATP to ADP. Thus, Hsp40 functions in a manner similar to
E. coli DnaJ (22).
Fig. 3.
Requirement of the J-domain and G/F region
for stimulation of the ATPase of Hsc70. Hsc70 (1.4 µM) and varying concentrations of His-Hsp40,
His-Hsp40(J+G/F) or Hsp40(J) were incubated with
[-32P]ATP (50 µM) for 30 min at
30 °C.
Fig. 4.
ATP-dependent stabilization of
Hsc70-peptide complexes by Hsp40. Hsc70 and
14C-peptide C were incubated for 30 min at 37 °C without
(lanes 1-3) or with Hsp40 (lanes 4-6) either in
the absence of nucleotide (
) or in the presence of ATP or ADP. The
Hsc70-peptide C complex was isolated on a Sephadex G-50 spin column.
The amounts of Hsc70 and bound peptide C were quantified as described
under ``Materials and Methods.'' Hsp40 alone did not bind peptide C
(data not shown).
Fig. 5.
Prevention of protein aggregation by Hsc70
and Hsp40 in the presence of ATP. A, luciferase was
incubated for 10 min at 42 °C in reaction mixtures containing the
indicated components followed by centrifugation at 16,000 × g for 5 min. Soluble and aggregated luciferase were analyzed
by SDS-PAGE and are presented as percent of total. B,
denatured rhodanese was diluted into reaction mixtures containing the
indicated combinations of Hsc70, Hsp40, and nucleotides. Aggregation
was monitored by turbidity at 320 nm for 10 min at room temperature and
is expressed as percent of the turbidity measured at 10 min in the
presence of ATP alone.
S were
without effect (Fig. 6B). Formation of a ternary complex was
also observed by size-exclusion chromatography on Sephacryl S-300; in
the presence of ATP, a substantial amount of luciferase cofractionated
with Hsc70 and Hsp40 as a high molecular mass complex larger than
thyroglobulin (Mr 670,000) (data not shown),
suggesting the presence of small aggregates of luciferase in
association with the chaperones. It is possible that in these complexes
Hsp40 interacts not only with Hsc70 but also with luciferase.
Fig. 6.
ATP-dependent complex formation
between Hsc70, Hsp40, and thermally denatured luciferase. A,
reaction mixtures of luciferase and the indicated combinations of
Hsc70, 6His-tagged Hsp40 (His-Hsp40), and ATP were incubated for 10 min
at 42 °C and then mixed with Ni2+-NTA-agarose. Retained
protein was eluted with 150 mM imidazole and analyzed by
SDS-PAGE. Luciferase protein was quantified by densitometry of
Coomassie stained gels. The amount of luciferase retained in the
presence of Hsc70, Hsp40, and ATP is set to 100%. B,
formation of a ternary complex was analyzed in the presence of ATP
(lane 1), ADP (lane 2), AMP-PNP (lane
3), and ATPS (lane 4). The amount of retained
luciferase in the presence of ATP is set to 100%.
Fig. 7.
-helices with a conserved tripeptide His-Pro-Asp
located in a solvent-accessible loop region (36, 37, 52). Substitutions
within this tripeptide abrogate the interaction with DnaK (38).
Interestingly, Hsp40 was unable to stimulate the ATPase of DnaK,
whereas E. coli DnaJ has the capacity to activate the
ATP-hydrolytic activity of mammalian Hsc70. The structural basis for
this differential behavior is not yet understood. The J-domain of Hsp40
alone was not sufficient to activate the ATPase of Hsc70. A similar
finding has been made for DnaK with the J-domain of DnaJ (38, 39). Only
when the J-domain was combined with the adjacent ~30 residue G/F
region could the ATP-hydrolysis stimulating activity be measured (this
study) (38, 39). On the other hand, an internal deletion of the G/F
region from DnaJ did not abolish its ATPase stimulating activity (50).
Furthermore, the NMR solution structures of the J-domain of DnaJ both
with and without the G/F region revealed no significant conformational
differences in the J-domain (36, 37). Thus, the G/F region may have a
more unspecific stabilizing effect on the J-domain, maintaining it in a
conformation suitable for the interaction with Hsc70.
Present address: Dept. of Cell Biology, The Tokyo Metropolitan
Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo
113, Japan.
¶
To whom correspondence should be addressed: Howard Hughes
Medical Institute and Cellular Biochemistry and Biophysics Program,
Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY
10021.
1
The abbreviations used are: PCR, polymerase
chain reaction; AMP-PNP, adenylylimidodiphosphate; ATPS,
adenosine-5-O-(3-thiotriphosphate); IPTG,
isopropyl-1-thio--D-galactoside; MOPS,
3-[N-morpholino]-2-hydroxypropanesulfonic acid; PAGE, polyacrylamide
gel electrophoresis; NTA, nitrilotriacetic acid.
2
Y. Minami and F.-U. Hartl, unpublished
results.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 G. J. Lee and E. Vierling A Small Heat Shock Protein Cooperates with Heat Shock Protein 70 Systems to Reactivate a Heat-Denatured Protein Plant Physiology, January 1, 2000; 122(1): 189 - 198. [Abstract] [Full Text] [PDF]
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A. A. Michels, B. Kanon, O. Bensaude, and H. H. Kampinga Heat Shock Protein (Hsp) 40 Mutants Inhibit Hsp70 in Mammalian Cells J. Biol. Chem., December 17, 1999; 274(51): 36757 - 36763. [Abstract] [Full Text] [PDF]
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P. J. Muchowski, L. G. Hays, J. R. Yates III, and J. I. Clark ATP and the Core "alpha -Crystallin" Domain of the Small Heat-shock Protein alpha B-crystallin J. Biol. Chem., October 15, 1999; 274(42): 30190 - 30195. [Abstract] [Full Text] [PDF]
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C. A. Ballinger, P. Connell, Y. Wu, Z. Hu, L. J. Thompson, L.-Y. Yin, and C. Patterson Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions Mol. Cell. Biol., June 1, 1999; 19(6): 4535 - 4545. [Abstract] [Full Text] [PDF]
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T. Laufen, M. P. Mayer, C. Beisel, D. Klostermeier, A. Mogk, J. Reinstein, and B. Bukau Mechanism of regulation of Hsp70 chaperones by DnaJ cochaperones PNAS, May 11, 1999; 96(10): 5452 - 5457. [Abstract] [Full Text] [PDF]
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 A. L. Fink Chaperone-Mediated Protein Folding Physiol Rev, April 1, 1999; 79(2): 425 - 449. [Abstract] [Full Text] [PDF]
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E. A. A. Nollen, J. F. Brunsting, H. Roelofsen, L. A. Weber, and H. H. Kampinga In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance Mol. Cell. Biol., March 1, 1999; 19(3): 2069 - 2079. [Abstract] [Full Text] [PDF]
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A. Carrello, E. Ingley, R. F. Minchin, S. Tsai, and T. Ratajczak The Common Tetratricopeptide Repeat Acceptor Site for Steroid Receptor-associated Immunophilins and Hop Is Located in the Dimerization Domain of Hsp90 J. Biol. Chem., January 29, 1999; 274(5): 2682 - 2689. [Abstract] [Full Text] [PDF]
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S. Takayama, Z. Xie, and J. C. Reed An Evolutionarily Conserved Family of Hsp70/Hsc70 Molecular Chaperone Regulators J. Biol. Chem., January 8, 1999; 274(2): 781 - 786. [Abstract] [Full Text] [PDF]
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 J. J. Silberg, K. G. Hoff, and L. E. Vickery The Hsc66-Hsc20 Chaperone System in Escherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System J. Bacteriol., December 15, 1998; 180(24): 6617 - 6624. [Abstract] [Full Text]
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J.-S. Liu, S.-R. Kuo, A. M. Makhov, D. M. Cyr, J. D. Griffith, T. R. Broker, and L. T. Chow Human Hsp70 and Hsp40 Chaperone Proteins Facilitate Human Papillomavirus-11 E1 Protein Binding to the Origin and Stimulate Cell-free DNA Replication J. Biol. Chem., November 13, 1998; 273(46): 30704 - 30712. [Abstract] [Full Text] [PDF]
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M. Gebauer, R. Melki, and U. Gehring The Chaperone Cofactor Hop/p60 Interacts with the Cytosolic Chaperonin-containing TCP-1 and Affects Its Nucleotide Exchange and Protein Folding Activities J. Biol. Chem., November 6, 1998; 273(45): 29475 - 29480. [Abstract] [Full Text] [PDF]
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Z. Lu and D. M. Cyr Protein Folding Activity of Hsp70 Is Modified Differentially by the Hsp40 Co-chaperones Sis1 and Ydj1 J. Biol. Chem., October 23, 1998; 273(43): 27824 - 27830. [Abstract] [Full Text] [PDF]
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Y.-G. Yeung, Y. Wang, D. B. Einstein, P. S. W. Lee, and E. R. Stanley Colony-stimulating Factor-1 Stimulates the Formation of Multimeric Cytosolic Complexes of Signaling Proteins and Cytoskeletal Components in Macrophages J. Biol. Chem., July 3, 1998; 273(27): 17128 - 17137. [Abstract] [Full Text] [PDF]
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J. Demand, J. Lüders, and J. Höhfeld The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors Mol. Cell. Biol., April 1, 1998; 18(4): 2023 - 2028. [Abstract] [Full Text]
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Z. Lu and D. M. Cyr The Conserved Carboxyl Terminus and Zinc Finger-like Domain of the Co-chaperone Ydj1 Assist Hsp70 in Protein Folding J. Biol. Chem., March 6, 1998; 273(10): 5970 - 5978. [Abstract] [Full Text] [PDF]
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A. A. Michels, B. Kanon, A. W. T. Konings, K. Ohtsuka, O. Bensaude, and H. H. Kampinga Hsp70 and Hsp40 Chaperone Activities in the Cytoplasm and the Nucleus of Mammalian Cells J. Biol. Chem., December 26, 1997; 272(52): 33283 - 33289. [Abstract] [Full Text] [PDF]
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L. H. Chamberlain and R. D. Burgoyne The Molecular Chaperone Function of the Secretory Vesicle Cysteine String Proteins J. Biol. Chem., December 12, 1997; 272(50): 31420 - 31426. [Abstract] [Full Text] [PDF]
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H. J. Oh, X. Chen, and J. R. Subjeck hsp110 Protects Heat-denatured Proteins and Confers Cellular Thermoresistance J. Biol. Chem., December 12, 1997; 272(50): 31636 - 31640. [Abstract] [Full Text] [PDF]
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K. Terada, M. Kanazawa, B. Bukau, and M. Mori The Human DnaJ Homologue dj2 Facilitates Mitochondrial Protein Import and Luciferase Refolding J. Cell Biol., December 1, 1997; 139(5): 1089 - 1095. [Abstract] [Full Text] [PDF]
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R.-F. Jiang, T. Greener, W. Barouch, L. Greene, and E. Eisenberg Interaction of Auxilin with the Molecular Chaperone, Hsc70 J. Biol. Chem., March 7, 1997; 272(10): 6141 - 6145. [Abstract] [Full Text] [PDF]
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Y. Morishima, P. J. M. Murphy, D.-P. Li, E. R. Sanchez, and W. B. Pratt Stepwise Assembly of a Glucocorticoid Receptor{middle dot}hsp90 Heterocomplex Resolves Two Sequential ATP-dependent Events Involving First hsp70 and Then hsp90 in Opening of the Steroid Binding Pocket J. Biol. Chem., June 9, 2000; 275(24): 18054 - 18060. [Abstract] [Full Text] [PDF]
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K. Terada and M. Mori Human DnaJ Homologs dj2 and dj3, and bag-1 Are Positive Cochaperones of hsc70 J. Biol. Chem., August 4, 2000; 275(32): 24728 - 24734. [Abstract] [Full Text] [PDF]
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D. Mamelak and C. Lingwood The ATPase Domain of hsp70 Possesses a Unique Binding Specificity for 3'-Sulfogalactolipids J. Biol. Chem., January 5, 2001; 276(1): 449 - 456. [Abstract] [Full Text] [PDF]
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E. A. A. Nollen, A. E. Kabakov, J. F. Brunsting, B. Kanon, J. Hohfeld, and H. H. Kampinga Modulation of in Vivo HSP70 Chaperone Activity by Hip and Bag-1 J. Biol. Chem., February 9, 2001; 276(7): 4677 - 4682. [Abstract] [Full Text] [PDF]
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P. J. Muchowski, G. Schaffar, A. Sittler, E. E. Wanker, M. K. Hayer-Hartl, and F. U. Hartl Hsp70 and Hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils PNAS, July 5, 2000; 97(14): 7841 - 7846. [Abstract] [Full Text] [PDF]
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E. A. A. Nollen, F. A. Salomons, J. F. Brunsting, J. J. L. v. d. Want, O. C. M. Sibon, and H. H. Kampinga Dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection PNAS, October 9, 2001; 98(21): 12038 - 12043. [Abstract] [Full Text] [PDF]
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