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INTRODUCTION |
Molecular chaperones of the Hsp70 family play a key role in the
control of protein folding during protein biogenesis, protein transport
through membranes, and when cells are exposed to proteotoxic stress
(1-3). Hsp70 chaperones have a specialized domain capable of binding
exposed hydrophobic stretches in polypeptide chains, which would
otherwise aggregate within the densely packed environment of the cell.
Substrate binding and release is coupled to a cycle of ATP binding and
hydrolysis by Hsp70. In vitro, a variety of regulatory
proteins of this Hsp70 cycle have been identified that influence the
properties of the Hsp70 chaperone in protein refolding.
Proteins of the Hsp40/DNA-J family can increase the Hsp70 chaperone
activity by enhancing the ATPase activity of Hsp70 (4, 5). This action
is dependent on the interaction of the J-domain of Hsp40 with the ATP
binding domain of Hsp70, but it also requires the interaction between
the extreme carboxyl-terminal EEVD sequence of Hsp70 and the
carboxyl-terminal domain of Hsp40. By using a luciferase reporter assay
in mammalian cells, we demonstrated that Hsp40 in vivo also
stimulates Hsp70-mediated refolding (6). In fact, by using dominant
negative Hsp40 mutants, we found that the Hsp70 chaperone activity was
completely inhibited, thereby proving that interaction with
J-domain-containing proteins is required for the in vivo
refolding activity of Hsp70 (7).
The homo-oligomeric protein Hip cooperates with Hsp70 in protein
folding by stabilizing the ADP-bound state of Hsp70. Hip directly binds
to the ATPase domain of Hsp70 when it is converted to the ADP-bound
state by proteins of the Hsp40 family (8). The Hsp70-binding site of
Hip consists of multiple tetratricopeptide repeats and a flanking
charged
-helix. The extreme amino terminus contains a domain
required for Hip oligomerization. As monomeric Hip still can bind to
Hsp70, homo-oligomerization is not required for Hsp70 interaction (9).
Besides affecting the Hsp70 chaperone cycle in vitro, Hip
alone can also bind to unfolded proteins and prevent their aggregation.
Yet refolding of proteins to their active state requires cooperation
with other chaperones (8, 10). The rat Hip protein shows >90%
identity at the amino acid level with p48, a protein that is involved
in the maturation of the human progesterone receptor (8, 11).
In addition to these positive regulators of the Hsp70 chaperone two
inhibitory proteins, CHIP and Bag-1, have been identified in
vitro. CHIP, also a tetratricopeptide repeat-containing protein, inhibits the ATPase activity of Hsp70 (12) and prevents the formation
of stable Hsp70-substrate interaction required for proper refolding.
Bag-1, originally discovered as a Bcl-2-associated protein (13), is
expressed in various isoforms, which originate from different
translation initiation sites, all of which can inhibit Hsp70
refolding activity both in vitro (14-16) and in
vivo (17). Recently several additional Bag-1-like proteins have
been identified (18). All Bag-1-like proteins share a conserved
40-50-amino acid "BAG" domain and compete with Hip in
binding to the ATPase domain of Hsp70 in vitro
(14-16, 18).
Here we investigated whether Hip and Bag-1 affect Hsp70 chaperone
activity in hamster fibroblasts and Chinese hamster ovary cells. In
both these cell lines, Hsc70 is constitutively expressed, whereas Hsp70
is only expressed upon exposure of the cells to stress. Overexpressed
Hip exclusively localized to the cytoplasm and enhanced refolding of
heat-inactivated cytoplasmic luciferase and protected it from
inactivation during ATP depletion. For both activities the presence of
the Hsp70-binding site as well as the oligomerization domain was
required. Hip was active when overexpressed alone as well as when
overexpressed together with Hsp70 suggesting that it stimulates the
action of both Hsc70 and Hsp70. As demonstrated before (17), Bag-1
inhibited Hsp70 chaperone function. In cells overexpressing Hip and
Bag-1, the inhibitory effects of Bag-1 were dominant. The results
reveal that in vivo a complex level of regulation of the
Hsp70 chaperone activity exists. Furthermore, they show that cycling
between an ATP/ADP-bound state is not absolutely required for the Hsp70
chaperone machine to be active.
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MATERIALS AND METHODS |
Plasmids and Constructs--
pRSVLL/V encodes cytoplasmic
luciferase under control of a Rous sarcoma virus-long terminal repeat
promoter (provided by Dr. S. Subramani, University of California, San
Diego). PSBC/Hip and pSBC vectors containing the various Hip mutants
were created by ligation of an EcoRI fragment from
pGAD10-Hip behind the SV40 promoter in pSBC-1 (provided by Dr. H. Hauser, Gesellschaft für Biotechnologische Forschung,
Braunschweig, Germany). Hip and Hip mutants were inserted into
pCDNA-3/HA1 after
polymerase chain reaction amplification resulting in an in-frame fusion
of a triple hemagglutinin tag.
pCDNA/HA-BAG-1 and pCDNA/HA-BAG-1
C were created by cloning
an EcoRI-XhoI fragment from pGEX-4T-1/BAG-1 and
pGEX-4T-1/BAG-1
C (14) behind a cytomegalovirus promoter in
pCDNA-3/HA (provided by Dr. S. Ness, Northwestern University,
Evanston, IL), respectively (17).
pCMV40 consists of a HindIII-BamHI fragment
encoding the cDNA sequence of human Hsp40 (19) inserted into the
BglII-HindIII sites of the eukaryotic expression
vector. Plasmid pCMV70 was constructed by inserting a
HindIII fragment encoding the cDNA for Hsp70.1, the
human-inducible DnaK homologue (20) into the HindIII site of
pCMV5 (6).
Cell Culture and Transfections--
OT70 cells are hamster lung
fibroblasts (O23) in which Hsp70 expression can be controlled by the
tetracycline-responsive transactivator (tTA) expression system
(21). Cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 1 mg/ml G418 (Life Technologies, Inc.), 1 mg/ml hygromycin (Roche
Molecular Biochemicals), and 3 µg/ml tetracycline (Sigma). G418 and
hygromycin were absent during all experiments.
For ATP depletion experiments Chinese hamster ovary cells (CHO) were
used as they were more sensitive to ATP depletion-mediated proteotoxicity than the hamster lung fibroblast. The CHO cells were
grown in Ham's F-12 medium supplemented with 10% fetal bovine serum
(Life Technologies, Inc.).
For both cell lines, transient transfections were performed using
LipofectAMINE according to the procedure of the manufacturer (Life
Technologies, Inc.).
Stress Treatments and Measurement of Luciferase
Activity--
For the heat shock experiments, OT70 cells were
transiently transfected with pRSVLL/V and co-chaperone-encoding
plasmids or pCDNA as a control. 24 h after transfection, the
cells were transferred into tissue culture tubes in medium with or
without 3 µg/ml tetracycline for induction of Hsp70 expression.
Another 24 h later, the medium was replaced with medium containing
20 µg/ml cycloheximide and 20 mM MOPS, pH 7.0. After a
30-min preincubation, cells were heated for 30 min at 45 °C to
inactivate luciferase. Subsequently, the cells were reincubated at
37 °C to allow for reactivation of luciferase. At various time
points, triplicate samples were taken for the measurements of
luciferase activity as described before (22).
For the ATP depletion experiments, CHO cells were transiently
transfected with pRSVLL/V and co-chaperone-encoding plasmids or
pCDNA as a control. 24 or 48 h after transfection, the cells were transferred into tissue culture tubes and incubated at 37 °C in
glucose-free Dulbecco's modified Eagle's medium in the presence of
3% fetal bovine serum, 10 mM 2-deoxyglucose (2-DG), and 20 µM carbonyl cyanide m-chlorophenylhydrazone
(CCCP) (23). During this incubation triplicate samples were taken for
measurement of luciferase activity.
Western Blot Analysis and Immunofluorescence Analysis--
For
Western blot analysis the cells were trypsinized, resuspended in
phosphate-buffered saline, lysed by addition of SDS-polyacrylamide gel
electrophoresis sample buffer, and sonicated prior to SDS/Western blot
analysis. The HA-tagged Hip and HA-tagged BAG-1 proteins were detected
using a monoclonal HA antibody (provided by Dr. R. Lamb, Northwestern
University, Evanston, IL). Hsp70 was detected by C92, a monoclonal
antibody specific for the heat-inducible form of Hsp70 (Stressgen).
Indirect immunofluorescence was performed as described previously (6).
OT70 cells transfected with pCDNA/HA-Hip or pCDNA/HA-BAG-1 were
immunostained with the mouse monoclonal anti-HA tag antibody and an
fluorescein isothiocyanate-labeled anti-mouse secondary antibody
(Sigma). The images were made by confocal laser scanning microscopy (Zeiss).
Measurement of Cellular ATP Levels--
Determination of the
relative ATP content in cell lysates was based on the same measurement
of ATP-dependent luciferase activity (22), whereas
exogenous luciferin and luciferase but not ATP were added to the
lysates (24). The light emission was measured on a luminometer for
10 s after mixing the cell lysate aliquots (0.15 ml) with 0.1 ml
of a lysing buffer containing 5 µg/ml luciferase, 0.3 mM
luciferin, 0.3 mM AMP, and 1 mM dithiothreitol.
The ATP level in untreated cells was considered as 100%. Care was
taken to use the same amount of cells (105) in the same
volume of a lysing buffer.
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RESULTS |
Overexpression of Hip Enhances Chaperone Capacity--
A
biochemical assay that allows the measurement of cellular chaperone
activity in both the cytoplasmic and nuclear compartment of mammalian
cells has been previously established (22). This assay has allowed us
to delineate the in vivo chaperone activity of Hsp70 and
modulation thereof by Hsp40/HdJ-1 (6, 7, 21) and Bag-1 (17). In this
model luciferase is expressed in the nucleus or cytoplasm of hamster
cells that are subsequently heated, after which activity of the enzyme
is measured at various time points after heat shock.
HA-tagged Hip was transiently expressed in OT70 hamster fibroblasts in
which only Hsc70 and not Hsp70 is constitutively expressed and in which
Hsp70 expression can be regulated by the tetracycline-responsive system
(21). Expression of HA-Hip did not influence basal Hsp70 nor did it
affect TET-inducible Hsp70 expression (Fig.
1A), as was shown before (8).
Hip was exclusively present in the cytoplasm (Fig. 1B) and
did not change its localization upon heat shock (data not shown). In
the absence of Hsp70 expression, overexpression of Hip resulted in an
increase in the refolding of the cytoplasmic luciferase (Fig.
1C). When overexpressed together with Hsp70, Hip also
enhanced the refolding of cytoplasmic luciferase to levels above that
for expression of Hsp70 alone (Fig. 1D). Consistent with the
cytoplasmic localization of Hip, no effect of Hip has been detected on
the inactivation and recovery of nuclear luciferase (data not
shown).

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Fig. 1.
Expression of Hip in OT70 enhances
Hsp70-mediated reactivation of heat-denatured firefly luciferase.
OT70 cells were transiently transfected with pCytluc (encoding
cytoplasmic luciferase) together with pCDNA (vector) or
pCDNA/Hip and grown in medium with or without tetracycline
(tet) for induction of Hsp70 expression. A,
Western blot analysis of Hsp70 and Hip expression. B,
immunofluorescent analysis of HA-Hip localization after staining the
transfected cells with a monoclonal HA antibody. C and
D, cells transfected with pCDNA (vector:
circles) or pCDNA/Hip (triangles) and grown
in medium with (C) or without (D) tetracycline
for regulated induction of Hsp70 expression were pretreated with
cycloheximide (20 µg/ml) and heated for 30 min at 45 °C to
inactivate luciferase. After heating, cells were allowed a recovery
period of 0-5 h at 37 °C, and samples for luciferase activity were
taken at the indicated time points. Data points represent the mean of 3 independent experiments and error bars indicate S.E. of the
means.
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To establish the mode of action of Hip-mediated enhancement of the
cellular chaperone capacity, we expressed a variety of mutant variants
that were previously characterized in vitro in terms of
their ability to bind to Hsc/Hsp70 and their ability to oligomerize
(Fig. 2, A and B
(9)). None of the expressed mutants resulted in an induction of Hsp70,
indicating that they did not cause a stress response in the cells.
Also, they did not affect the level of induction of Hsp70 by
tetracycline removal (data not shown), and all mutant proteins were
expressed to equal levels as the wild-type proteins as detected by an
HA antibody (Fig. 2B). The Hip mutant with a truncation of
the homo-oligomerization domain but containing the entire Hsp70 binding
domain (Hip-(15-368)) was not capable of stimulating luciferase
refolding after heat shock like wild-type Hip alone (Fig.
2C) and also could not stimulate refolding in the presence
of Hsp70 (Fig. 2D). This implies that oligomerization of Hip
is required for its enhancement of the cellular chaperone activity.
Expression of the mutant Hip that lacks part of or the entire Hsp70
binding domain but contains the amino-terminal oligomerization domain
(Hip-(1-218)) was also incapable of stimulating protein folding
in the cell (Fig. 2, C and D). Thus, the observed
enhanced refolding under the condition of Hip overexpression seems to
depend on the interaction with Hsp70 or Hsc70 (when the
tetracycline system is not induced). Expression of the
Hip-(110-368) protein, lacking both the Hsp70 binding and
oligomerization capacity was also ineffective in stimulating Hsp70-mediated recovery (Fig. 2, C and D).
However, the mutants Hip-(1-303) and Hip-(1-257) were
as effective as the full-length Hip in enhancing luciferase refolding
(Fig. 2, C and D). This indicates that the last
110 carboxyl-terminal amino acids (amino acids 258-368) are not
essential in for the effects of Hip.

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Fig. 2.
The Hsp70 binding domain and oligomerization
domain of Hip are required for enhancement of Hsp70-mediated
reactivation of heat-denatured firefly luciferase. OT70 cells were
transiently transfected with pCytluc (encoding cytoplasmic luciferase)
together with pCDNA (vector) or the indicated full-length
(wt) or mutant Hip cDNAs and grown in medium with or
without tetracycline (tet) for induction of Hsp70
expression. A, constructs used and their in vitro
characteristics (after Irmer and Hohfeld (9)). B, Western
blot analysis of mutant Hip expression (HA-Hip bands are indicated by
white arrowheads). C and D,
transfected cells were pretreated with cycloheximide (20 µg/ml) and
heated for 30 min at 45 °C to inactivate luciferase. After heating,
cells were allowed a recovery period of 3 h at 37 °C, and samples
for luciferase activity were taken at the indicated time points. Data
points represent the mean of 3 independent experiments, and error
bars indicate S.E. of the means.
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Effects of Hsp70 and Hip Under Conditions of ATP Depletion--
By
having established that Hip can stimulate Hsp70-mediated refolding of
heat-inactivated luciferase, we wanted to gain further insight in the
mechanism of this effect. In vitro data have shown that Hip
results in the stabilization of the Hsp70-ADP substrate complex (8). To
test this in vivo we decided to investigate the effect of
Hsp70 and Hip in cells after ATP depletion. Such treatments were shown
to be proteotoxic and capable of inducing protein inactivation and
aggregation, including inactivation and insolubilization of the
reporter enzyme firefly luciferase (24-26). Here, ATP depletion in CHO
cells was accomplished by treatment with 10 mM
2-deoxyglucose + 20 µM CCCP in the glucose-free medium. This treatment results in a rapid decline of total cellular ATP to less
than 2% after 60 min (Fig.
3A). Overexpression of
chaperones had no effect of the kinetics of ATP depletion (data not
shown).

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Fig. 3.
Hsp70-mediated protection of luciferase
inactivation during ATP depletion: effects of Hip. CHO cells were
transiently transfected with pCytluc (encoding cytoplasmic luciferase)
alone or together with cDNAs encoding the various (co)chaperones
and treated with CCCP + 2-DG 48 h after transfection.
A, kinetics of ATP depletion (circles) and
luciferase inactivation (squares) during treatment with CCCP + 2-DG. B, effects of overexpression of Hsp70
(triangles), Hsp40 (squares), or Hsp70 + Hsp40
(diamonds) on luciferase inactivation during ATP depletion
(circles: vector only). C, effects of
overexpression of either full-length Hip (wild type (wt),
triangles) or mutant Hip cDNA (closed
squares, Hip-(1-219); diamonds, Hip-(15-368);
open squares, Hip-(110-368)) on luciferase inactivation
during ATP depletion (circles, vector only).
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As a result of the ATP depletion luciferase activity gradually declined
in the cells, with kinetics about 4-fold slower than that for the ATP
depletion (Fig. 3A). In contrast to the situation after heat
shock where luciferase can be refolded from the heat-denatured state
when the heat treatment is not severe or when chaperones are
overexpressed, no significant luciferase refolding was observed upon
ATP replenishment under any of the conditions tested (data not shown).
Remarkably, when the ATP-dependent Hsp70 chaperone was
overexpressed, the rate of luciferase denaturation during ATP depletion
was clearly attenuated. Hsp40/Hdj-1 overexpression alone or combined
with Hsp70 had no effect on the rate of luciferase inactivation during
ATP depletion (Fig. 3B). The latter is consistent with the
presumed mechanistic action of Hsp40, i.e. stimulation of
the conversion of the ATP-bound state of Hsp70 to an ADP-bound state by
accelerating ATP hydrolysis (4, 5).
Like for heat shock, full-length Hip was capable of enhancing the
cellular chaperone activity under conditions of ATP depletion. Luciferase inactivation was retarded in cells overexpressing Hip; this
action was again dependent on the ability of Hip to oligomerize and to
bind to Hsp/Hsc70 as mutants lacking the respective domains were
incapable of demonstrating chaperone activity (Fig. 3C). Combining Hip and Hsp70 overexpression led to a moderate further protection compared with either Hip or Hsp70 overexpression alone (data
not shown).
Bag-1 Suppresses Hip-mediated Enhancement of the Hsp70 Chaperone
Function--
We have previously shown that various isoforms of the
Bag-1 protein can inhibit the in vivo chaperone action of
Hsp70 at physiologically relevant, stoichiometric levels (17). In
vitro data have indicated that both Hip and Bag-1 compete for
interaction with the ATPase domain of Hsp70 in vitro
(14-16, 18).
Therefore, we transiently coexpressed Bag-1 (29-kDa isoform) and Hip,
either alone or jointly in luciferase expressing OT70 cells, and we
examined the effects of Hsp70-mediated reactivation of heat-inactivated
luciferase. As demonstrated before (17) the 29-kDa Bag-1 predominantly
localized in the nucleus but also could be clearly detected in the
cytoplasm (Fig. 4A). Both Hip and Bag-1 were HA-tagged and expressed to equal total levels in OT70
cells (Fig. 4B). Considering the exclusive cytoplasmic
localization of Hip (Fig. 1B), we therefore conclude that
its cytoplasmic concentration is higher than the cytoplasmic
concentration of Bag-1. The HA-Bag-1, although predominantly nuclear,
was capable of substantially inhibiting the Hsp70-mediated recovery of
cytoplasmic luciferase. Surprisingly, coexpression of Bag-1 and Hip led
to one-half the reactivation of the cytoplasmic luciferase compared
with overexpression of Hip and Hsp70 alone (Fig. 3C). This
suggests that the inhibitory effect of Bag-1 is dominant to the
stimulatory effect of Hip. The inhibitory effect of Bag-1 requires its
carboxyl-terminal Hsp70 binding domain as overexpression of Bag-
C
did not inhibit the stimulatory effects of Hip (data not shown).

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Fig. 4.
Dominant inhibition of the Hip-mediated
enhancement of the Hsp70 chaperone activity by Bag-1. OT70 cells
were transiently transfected with pCytluc (encoding cytoplasmic
luciferase) together with pCDNA (vector) or pCDNA/Hip,
pCDNA/Bag-1 or pCDNA/Hip, and pCDNA/Bag-1 together. The
cells were grown in medium with or without tetracycline
(tet) for induction of Hsp70 expression. A,
immunofluorescent analysis of HA-Bag-1 localization after staining the
transfected cells with a monoclonal HA antibody. B, Western
blot analysis of Hsp70, Hip, and Bag-1 expression. C, cells
transfected with pCDNA (vector, circles), pCDNA/Hip
(triangles), pCDNA/Bag-1 (squares), or
pCDNA/Hip and pCDNA/Bag-1 (diamonds) were grown in
medium without tetracycline (tet) for induction of Hsp70
expression. After pretreatment with cycloheximide (20 µg/ml), the
cells were heated for 30 min at 45 °C to inactivate luciferase and
allowed a recovery period of 0-5 h at 37 °C. Samples for luciferase
activity were taken at the indicated time points. Data points represent
the mean of 3 independent experiments, and error bars
indicate S.E. of the means.
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DISCUSSION |
In vitro experiments have identified several partner
proteins of Hsp70 that regulate its function as a molecular chaperone. These include both positive regulators (Hsp40/Hdj1 and Hip) and negative regulators (CHIP, Bag-1) (4, 8, 12, 14-16, 18). One valid
issue is whether in vitro chaperone-mediated protein refolding studies accurately reflect in vivo events given
the high cellular concentration of proteins, the vast excess of
potential substrates, and that each cellular compartment has multiple
chaperones. Here, we have focused on the in vivo action of
Hip, a member of a distinct family of Hsp70 co-chaperones that
interacts with the Hsp70 ATPase domain and enhances folding of
denatured luciferase in vitro (8, 27).
In vivo, Hip is a component of steroid hormone receptor
complexes and is also involved in the assembly of Hsp70 into
multichaperone complexes with Hsp90 (11, 28). A role for Hip as a
positive regulator of Hsp70 activity in vivo had not been
demonstrated previously. Both constitutively expressed Hip as well as
overexpressed Hip was found to be exclusively located in the cytoplasm
(Ref. 8 and this report). This localization did not change during or
after heat shock (not shown), unlike Hsp70 and Hsp40 that change their
localization from mostly cytoplasmic before to merely nuclear after
heat shock (29-32). Consistent with its cytoplasmic localization, Hip
enhanced refolding of the cytoplasmic but not nuclear luciferase. The
effect was already seen when Hip was overexpressed alone, and
coexpression with Hsp70 merely resulted in additive enhancement of
luciferase refolding.
In vitro, Hip was shown to be capable of binding directly to
unfolded proteins and to prevent their aggregation (8, 10). As such,
one could interpret the increase in cellular chaperone activity by Hip
overexpression independent from Hsp70. However, deletion of the
Hsp70-binding site of Hip without affecting its homo-oligomerization
domain resulted in the loss of a Hip effect on luciferase refolding
after heat shock. Although this mutant was never tested for its ability
to prevent aggregation in vitro, the most likely explanation
for observed effects of Hip in mammalian cells is that Hip requires a
direct interaction with Hsp70 consistent with the situation in
vitro (8, 10). Furthermore, this is supported by the finding that
Bag-1, which competes with Hip for Hsp70 binding (see also below),
inhibited the Hip-mediated increment in cellular chaperone activity.
Since Hip also stimulated refolding in the absence of Hsp70 expression,
it can be deduced that Hip is at least equally effective in stimulating
refolding by Hsc70, which is constitutively expressed in the OT70 cells.
By using a series of other Hip mutants, it was demonstrated that Hip
oligomerization is also required for its action as a co-chaperone of
Hsp70/Hsc70, whereas the last 110 carboxyl-terminal amino acids are not
essential for this function. The amino acids 278-311 of Hip contain 7 imperfect GGMP repeats that are found in most cytoplasmic Hsc70s but
not in Hsp70s (33). The function of this motif is yet unclear, but from
our in vivo data as well as from in vitro data
(9) it can be deduced that it is not involved in regulating Hsp70
chaperone activity. Furthermore, our data indicate that the very
carboxyl-terminal domain (amino acids 316-368), which is structurally
related to the Hop homologues Sti1p and human IEF SSP3521, is not
required for regulating Hsp70 refolding activity.
Our data on effects of chaperones on ATP depletion-induced protein
damage revealed several new features. It seems plausible to assume that
most if not all Hsp70s will be in the ADP-bound state under conditions
of ATP depletion. This notion is supported by the fact that we could
not detect a stimulating effect of Hsp40/Hdj-1 on the chaperone
activity of Hsp70 in ATP-depleted cells. Hence, our data indicate that
the ADP-bound Hsp70 can still act as a chaperone despite its lower
substrate on rate (34). The data also imply that Hsp70 does not need to
undergo cycling between the ATP and ADP states to protect against
protein inactivation. This is consistent with the observation that
mutant Hsp70s that lack their entire ATPase domain are still capable of
protecting against proteotoxic damage (35). Finally, the finding that
Hip is capable of stimulating the chaperone activity of Hsp70 under ATP-depleted conditions is consistent with the suggestion that Hip
stabilizes the interaction of substrate with Hsp70 in its ADP-bound
state. Indeed in vitro data showed that Hsc70 only binds to
Hip immobilized on Ni2+-nitrilotriacetic acid-agarose
columns when preincubated with ADP but not ATP (8). Also, Hip cannot
associate with the Hsc70 mutant K71E, a mutant that cannot hydrolyze
ATP (11, 28). For the in vivo situation, these data imply
Hsp70 and Hip may be functional to combat the protein damage as it
occurs during ischemic insults where cellular ATP levels severely drop.
As both Hip and BAG-1 interact with the Hsp70 ATPase domain, we finally
examined whether and how they may functionally compete in regulating
the in vivo chaperone activity of Hsp70. In vitro data had already shown that Bag-1 binding to Hsp70 results in a
displacement of the Hsp70 cofactors Hsp40, Hop, and Hip (15, 16, 36,
37) indicating that it acts dominantly over these other regulators.
Indeed, we already demonstrated previously that physiological increases
in the cellular level of Bag-1 inhibit the Hsp70-dependent
chaperone activity in cells that constitutively express Hsp40 and Hip.
This activity was seen for multiple isoforms of Bag-1 with molecular
masses of 29, 33, 46, and 50 kDa that are expressed in cells as
originating from different start codons (17). Here we used the 29-kDa
isoform that is predominantly located in the nucleus but showed
cytoplasmic localization and was capable of inhibiting the refolding of
the heat-inactivated cytoplasmic luciferase (17). By using HA-tagged
proteins, we could transfect transiently with Bag-1 and Hip such that
equal total levels of the proteins were expressed. With Hip being
exclusively cytoplasmic, we therefore concluded that it was present in
the cytoplasm in excess over Bag-1. Still, under these conditions we
observed that Bag-1 could strongly diminish the refolding of heat-denatured cytoplasmic luciferase in the presence of excess Hip. So
in vivo, like in vitro, Hsp70 prefers association
with Bag-1 in the presence of Hip, and we conclude that the effects of
positive and negative regulators of Hsp70 can be reproduced in both
systems yielding the same results.
To what extent can we draw conclusions on the regulation of Hsp70
activity in dealing with stress-induced protein damage by overexpression of co-chaperones? Neither Hip nor BAG-1 are classical heat shock proteins as their levels are not induced in stressed cells,
whereas Hsp70 and Hdj-1/Hsp40 are strongly induced and accumulate to
high levels following heat shock or ATP depletion. Furthermore, the
relative ratio of Hsp70 and co-chaperones changes within the cell due
to the heat shock-induced translocation of Hsp70 and Hsp40 to the
nucleus (29-32), whereas the localization of Hip and Bag-1 remains
unchanged (Ref. 7 and data not shown). We previously showed that in
hamster lung fibroblasts (O23 cells) elevated Hsp70 levels alone seemed
to be sufficient to provide resistance toward cytoplasmic
proteotoxicity comparable to that observed in pre-stressed cells. For
the nuclear chaperone activity other factors seem to be required in
addition to an increase in the level of Hsp70 to provide resistance
comparable to that observed in pre-stressed O23 cells (21). However,
under normal conditions of cell growth and differentiation, the levels
of Hip and Bag-1 vary among different tissue culture cell lines and
tissues (38). This suggests that the ultimate Hsp70 chaperone function
both before and after stress could vary not only according to the total expression levels of Hsp70 but also to the levels of expression of the
positive or negative co-chaperones, their affinity toward Hsp70, their
cell-organelle-specific localization, and condition-mediated (re)distributions of the (co)chaperones.