From the Laboratory of Molecular Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255
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ABSTRACT |
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The heat shock transcription factor (HSF)
mediates the induction of heat shock gene expression. The activation of
HSF involves heat shock-induced trimerization, binding to its cognate
DNA sites, and the acquisition of transcriptional competence. In this
study, the oligomeric properties of Drosophila HSF were
analyzed by equilibrium analytical ultracentrifugation and gel
filtration chromatography. Previous findings showed that trimerization
of purified Drosophila HSF was directly sensitive to heat
and oxidation (1). Here we report that low pH, in the physiological
range, also directly induces HSF trimerization and DNA binding in
vitro. Furthermore, the induction of HSF trimerization by low pH
is synergistic with the actions of heat and oxidation. Since heat or
chemical stress leads to a moderate decrease of intracellular pH, we
suggest that intracellular acidification may contribute to activating
the heat shock response in vivo.
All living organisms respond to elevated temperature and a variety
of chemical treatments by a rapid increase in the synthesis of heat
shock proteins. These protect the structure and activity of proteins
from denaturation under environmental stresses (2-5). In eukaryotes,
transcriptional regulation of heat shock genes is mediated by a
pre-existing transcription activator (heat shock factor,
HSF1), which binds to an
upstream conserved DNA recognition sequence (heat shock element, HSE)
and acquires transcriptional potency, leading to an increase in the
synthesis of heat shock proteins (6-9). HSF is encoded by a
single-copy gene in some species and by several genes in others, but
only one HSF, designated HSF1, appears to be sensitive to the heat
stress signal. In metazoans, HSF is present in an inactive monomeric
form under normal conditions and converts to a trimeric form with high
DNA binding affinity after stress induction. The activated HSF trimer
also gains transcriptional competence and undergoes
hyperphosphorylation. Under certain conditions, the DNA binding
activity of HSF can be uncoupled from the acquisition of
transcriptional activity (9).
All HSFs contain two highly conserved regions: a DNA-binding domain at
the amino terminus and an adjacent trimerization domain with clusters
of hydrophobic heptad repeats (HR-A/B) separated by a short spacer (9).
The DNA-binding domain of HSF forms a compact structure resembling the
helix-turn-helix DNA-binding motif (10, 11). The trimerization domain
has been proposed to resemble the hemagglutinin trimer of influenza
virus, where the long array of heptad repeats self-associates as a
parallel triple-stranded, The activation of HSF involves complex signaling pathways, including
the loss of feedback repression imposed by the constitutive HSP70
proteins (25-30), serine phosphorylation of HSF (31-37), and direct
effects of temperature (38-43). To test these mechanisms, we
established an in vitro assay for reversible trimerization and DNA binding of Drosophila HSF purified from nonshocked
or heat-shocked Sf9 cells (1). This in vitro system
enabled analysis of DNA binding and the equilibrium distributions of
monomer and trimer forms of HSF in vitro under a variety of
inducing conditions. Using this system, we observed direct and
reversible effects of heat and oxidation on HSF trimerization and DNA
binding activity, revealing a physico-chemical mechanism for signal
transduction of the heat shock response (1). However, the changes in
trimerization and DNA binding induced by heat and hydrogen peroxide
treatments in vitro were not as pronounced as those observed
after heat shock in cells (1). This prompted us to search for
additional conditions that could further enhance HSF activation
in vitro. Here, we report that moderately low pH in the
physiological range directly and reversibly induces trimerization and
DNA binding of purified Drosophila HSF. Such a change of pH
may play an additional regulatory role in heat shock response in
vivo.
Protein Purification--
The Drosophila HSFs were
expressed in Sf9 (Spodoptera frugiperda) cells
infected with baculovirus (multiplicity of infection of 5-10) carrying
the Drosophila hsf cDNA inserted with no additional residues in pBlueBac (Invitrogen) (44). Whole cell extracts were
prepared from either the nonshocked or heat-shocked (36 °C, 30 min)
Sf9 cells, and HSFs were purified to 95% homogeneity by using
heparin Sepharose CL-6B, HSE-agarose, and Mono S chromatography (1).
The purity of HSF protein was estimated by reverse phase chromatography
and SDS-polyacylamide gel electrophoresis. Absorption spectroscopy was
used to determine the concentration of HSF. The extinction coefficient
of HSF, 35970 M Analytical Ultracentrifugation--
Equilibrium analytical
ultracentrifugation of HSFs was performed using a Beckman XLA
analytical ultracentrifuge at a rotor speed of 12,000 rpm at 20 °C.
The buffer used was in 20 mM sodium phosphate (pH 6.6 or
7.4), 150 mM KCl, 1 mM MgCl2, 0.2 mM DTT, and 5% glycerol (v/v). Equilibrium was considered
to have been attained when the absorbance scans at 230 nm were
invariant for 12 h. The models for fitting the absorbance data
matrix are: monomer only, A230,r =
Ab·exp( Gel Filtration Chromatography--
Precision columns (PC 3.2/30,
2.4 ml) and a SMART system (Amersham Pharmacia Biotech) were used for
gel filtration analysis. 0.5 µM purified HSF samples (in
30 µl) were fractionated on a Superose 6 column equilibrated in
buffer containing 20 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 200 mM KCl, 0.5 mM DTT, and 5% glycerol at a flow rate of 40 µl/min and
8 °C. This low temperature was utilized to minimize trimer
dissociation upon sample dilution during the 1-h chromatography; we
have shown previously that trimer dissociation is time- and
temperature-dependent (1). Thyroglobulin (670 kDa),
Gel Mobility Shift Assay and Western Blotting Analysis--
Gel
mobility shift assays of Drosophila HSF were according to
Zhong et al. (44), using 32P-5'-end-labeled
double-stranded oligonucleotides corresponding to a HSE. After
induction, about 30 ng of HSF was mixed with labeled HSE and held on
ice before agarose gel electrophoresis at room temperature. Western
blotting was performed as described previously (48), using polyclonal
antibodies against Drosophila HSF and enhanced
chemiluminescent detection (ECL, Amersham Pharmacia Biotech). Typically, 10-20 ng of HSF was analyzed per sample.
Intracellular pHi Measurements--
SL2 cells were
grown at room temperature (22-24 °C) in spinner culture with
Schneider's medium (Life Technologies, Inc.) supplemented with 10%
heat-inactivated fetal bovine serum and 25 µg/ml gentamicin. Cell
suspensions (typically 3-5 × 106 cells/ml) were
centrifuged, washed, and resuspended in S-medium containing 25 mM KCl, 5 mM NaHCO3, 15 mM MgSO4, 5 mM CaCl2,
20 mM glucose, 100 mM NaCl, and 10 mM PIPES/Tris (pH 6.8-6.9). This solution was designed to
approximate the ionic composition of Schneider's medium (49). Cells
were then incubated with 5 µM 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester
(BCECF/AM, Molecular Probes) in S-medium (~1.5 × 107 cells/ml) for 30 min at 23 °C with constant shaking
at 200 rpm. At the end of the incubation, cells were washed twice with
S-medium. Aliquots of cells were centrifuged, resuspended at ~2 × 106 cells/ml in S-medium in the presence or absence of
chemical inducers. At room temperature, cells remained viable with
little leakage of BCECF for several hours. The BCECF fluorescence was
monitored with a SLM8000C spectrofluorometer as the ratio of emission
at 530 nm for excitation at 500 nm, to the emission upon excitation at
450 nm (slit width 2 nm). The fluorescence signal was calibrated with
the nigericin-high K+ technique (50). Briefly, cells were
suspended in a series of solutions containing 150 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 2 µM nigericin (Molecular Probes) and 10 mM
MES/Tris (pH range 5.5-6.0) or PIPES/Tris (pH range 6.2-7.8). The
calibration curves were fitted by polynomial regression of degree 3 [F500/F450 = a + b(pH) + c(pH)2 + d(pH)3], and pHi values were calculated
using the fitted parameters.
Low pH Induces HSF Trimerization in Vitro--
Several lines of
evidence have previously suggested that there is a change in
intracellular pH during heat shock of living cells (51-54). A
substantial increase in DNA binding of HSF was also reported when crude
extracts were subjected to moderately acidic pH conditions (pH 6.0)
(42, 43). To test whether low pH could induce the change in the
oligomeric state of HSF directly, or if the pH signal was sensed and
transduced by other factors present in the crude extracts, we analyzed
the oligomeric states of purified Drosophila HSF as a
function of pH. The Drosophila HSF used was overexpressed in
either nonshocked or heat-shocked Sf9 cells and purified by
chromatography to 95% homogeneity. Fig. 1A shows the pH dependence of
the monomer-trimer equilibrium of HSF as measured by gel filtration
chromatography. At 0.5 µM HSF concentration and 20 °C,
the HSF trimer population showed a sigmoidal increase from ~26% to
~78% over a pH drop from pH 8.3 to pH 5.6, with the greatest
increase occurring in the range of pH 7.5 to pH 6.5. Importantly, the
induction of HSF trimerization by the pH decrease was reversible. When
the HSF sample in pH 6.6 buffer was re-equilibrated in pH 7.4 buffer,
the percentage of trimers decreased from ~63% to the original level,
~40% (Fig. 1B). This reversibility indicates that the
observed changes in state of HSF are unlikely to be due to irreversible
effects of acidic pH on the activity of misfolded protein. We conclude
that a moderate decrease of pH is able to make a direct contribution to
HSF trimerization.
Equilibrium Analysis of HSF Oligomerization by Analytical
Ultracentrifugation--
In addition to gel filtration chromatography,
the oligomeric states of purified HSFs were measured by analytical
ultracentrifugation, which is rigorously based upon the reversible
thermodynamics of protein-protein association. As shown in Fig.
2, the equilibrium concentration
distribution of HSF at 230 nm could not be fitted as a homogeneous
monomer, dimer, or trimer but did fit well to a monomer-trimer
equilibrium. Western blotting also shows no significant protein
degradation when samples were analyzed after equilibrium sedimentation
at 20 °C for 72 h (Fig. 2 (inset)). The
sedimentation data confirm that purified Drosophila HSF is
distributed as a mixture of monomers and trimers at submicromolar
concentration, neutral pH (7.4), and room temperature (20 °C).
We were able to derive the equilibrium dissociation constant
(Kd) of HSF from analytical ultracentrifugation at
pH 7.4 and compare this value with the Kd for HSF at
pH 6.6. As shown in Table I, a drop from
pH 7.4 to pH 6.6 led to a 10-fold decrease in the Kd
(i.e. increased trimerization). While the
Kd values derived from gel filtration chromatography are generally 2-3-fold larger than those from analytical
centrifugation (probably due to trimer dissociation during
chromatography), the magnitude of the change in Kd
values induced by a pH drop from 7.4 to 6.6 is nonetheless similar for
both techniques. Interestingly, as was shown by gel filtration (1), and
confirmed by analytical ultracentrifugation (Table I), no significant
difference of the Kd values were observed for
nonshocked and heat-shocked HSF, indicating that covalent modifications
of HSF induced by heat shock in this expression system have no effect
on trimer formation.
Low pH Activates DNA Binding of HSF in Vitro--
The effect of
low pH on the DNA binding of HSF at 20 °C was further investigated
using the gel mobility shift assay. As shown in Fig.
3, both purified nonshocked and
heat-shocked HSF showed enhanced DNA binding activity when the buffer
pH was decreased, consistent with increased HSF trimerization. The
maximal DNA binding of HSF at 20 °C occurred at pH 7.0 (Fig. 3,
lanes c and h). The magnitude of the
increase in HSF DNA binding activity caused by lowering the pH from 7.4 to 7.0 was comparable with the increase caused by heat treatment alone
(Fig. 3, lanes b and g).
Interestingly, a further drop of buffer pH to 6.2 led to a decrease of
DNA binding activity (Fig. 3, lanes e and
j); this effect is likely to be due to partial denaturation
of HSF brought about by increasingly acidic pH.
Synergistic Regulation of Drosophila HSF Trimerization by Low pH,
Heat, and Oxidation--
Previously, we identified heat and oxidation
as two direct inducers of HSF trimerization and DNA binding in
vitro. Each individual inducing signal decreases the
Kd of Drosophila HSF by 8~11-fold,
leading to a 30~40% increase of the HSF trimer population at
physiological concentrations (1). To evaluate if low pH could act
synergistically with heat and/or oxidation, we measured the
Kd values of HSF under multiple inducing conditions using gel filtration chromatography. As shown in Table
II, treatments with
heat/H2O2, heat/low pH (pH 6.6) or
H2O2/low pH (pH 6.6) decreased the
Kd values of HSF by about 30.6-, 25.0-, and
20.4-fold, respectively, somewhat more than the sum of individual
changes. In a triple induction by heat, H2O2
and low pH (pH 6.6), the Kd value of HSF decreased
by 45.8-fold (Table II), increasing the HSF trimer population by
50~60%. The synergistic effect of low pH on heat-induced HSF
trimerization in vitro may have a role in the heat shock
response in vivo.
Intracellular Acidification by Heat and Other Inducers--
To
investigate the physiological significance of the low pH-induced
trimerization and DNA binding of HSF, we measured the intracellular pH
(pHi) change in Drosophila SL2 cells during heat or
chemical stresses, using the fluorescent indicator BCECF as a marker of
the intracellular pH (55). SL2 cells were grown in spinner culture with
Schneider's medium (pH 6.8) at 22-24 °C. The fluorescence signal
was calibrated using the nigericin-high K+ technique (50).
After exposure of the BCECF-loaded cells to a series of solutions (pH
ranging from 5.5 to 7.8) in the presence of nigericin, the ratio of
fluorescence intensities (at 530 nm) was measured for the excitation
wavelengths of 500 and 450 nm. Fig.
4A shows the BCECF
fluorescence ratio as a function of intracellular pH. Data were fitted
with a polynomial regression of degree three; the fitted parameters
were then used to convert fluorescence ratios to pHi
values.
As shown in Fig. 4B, the resting pHi of SL2 cells
under normal conditions was 7.36 ± 0.02 at 23 °C. After heat
shock for 10 min at 37 °C, the pHi dropped to 7.05 ± 0.02. The magnitude of pHi change in SL2 cells is consistent with the pHi decrease reported previously in salivary glands
upon heat shock (from pH 7.38 to 6.91) (51). In addition to heat shock
(37 °C, 10 min), chemical inducers of the heat shock response such
as ethanol, sodium salicylate, arachidonate, and indomethacin (group I
inducers) lowered the pHi of SL2 cells by 0.15-0.36 units
(Table III). Interestingly, sodium
arsenite, cadmium sulfate, and canavanine (group II heat shock
inducers) had no significant effects on the intracellular pH value
(Table III). Among the group I inducers, heat shock and sodium
salicylate have been shown to rapidly activate HSF DNA binding, induce
chromosomal puffing or HSP synthesis in Drosophila (56, 57),
and ethanol, arachidonate, and indomethacin are fast heat shock
inducers or potentiators in mammals (58-60). In contrast, it is known
that the group II chemicals such as cadmium sulfate and sodium arsenite are not very good inducers for HSP synthesis in SL2 cells, and the
induction of heat shock proteins by canavanine is a slow process in
Drosophila (61, 62). Therefore, the rapid drop in
intracellular pH by some heat shock inducers detected in our
measurements generally correlates well with the kinetics of induction
of HSF DNA binding or HSP synthesis (with the exception of hydrogen
peroxide). Taken together, our data suggest that a drop in
intracellular pHi may contribute to the induction of HSF
trimerization in vivo by a subset of heat shock
inducers.
In this study, we demonstrate that low pH in the physiological
range of pH 7.5 to 6.5 can directly and reversibly induce trimerization and DNA binding of purified Drosophila HSF in
vitro. This in vitro sensitivity of
Drosophila HSF to pH is generally consistent with earlier
reports showing that the DNA binding of HSF can be induced by low pH in
crude human and Drosophila cell extracts (42, 43) and also
with a recent study showing pH effects on bacterially expressed mouse
HSF1 protein (38). We note, however, that the latter effects on mouse
HSF1 were not shown to be reversible and that a more acidic pH was
required (maximal effect observed at pH 5.9) (38). We show further that
the direct effects of low pH are synergistic with heat and oxidation,
leading to a 30-40-fold decrease in the Kd for
trimer dissociation of purified HSF. As such, the combined effects of
low pH and heat on HSF can account for a substantial portion, though
not all, of the induction of trimerization and DNA binding activity of
HSF observed in cells. Since both heat stress and a number of chemical
inducers of the heat shock response lead to a decrease of the
intracellular pH in living cells, our results suggest that moderate
intracellular acidification may contribute to the induction of HSF
activity during heat shock in vivo by several inducers of
the response.
How are the effects of low pH transduced to HSF? Many proteins are
known to undergo a conformational change in the process of activation.
Influenza virus hemagglutinin, for example, undergoes a low pH-induced
conformational change that mediates fusion of the viral and host cell
membrane (63). This conformational change of hemagglutinin protein,
irreversible by nature, can also be triggered by heat or treatment with
urea in vitro (64). Interestingly, the pH effects on HSF
activation are reversible, indicating that change in the state of HSF
is driven by thermodynamic rather than kinetic considerations. Previous
studies have established that trimerization of HSF is subject to
negative regulation and requires the integrity of both amino- and
carboxyl-terminal hydrophobic heptad repeats, in addition to other
regions of the molecule (18-20). These findings have led to a model
for trimer regulation whereby the carboxyl-terminal domain of HSF is
involved in the suppression of trimerization of the amino-terminal
heptad repeats. There are two histidine residues and more than 20 other
charged amino acids located in the middle of trimerization domain
(between HR-A and HR-B) and in the carboxyl-terminal HR-C, which are
conserved in all HSFs or conserved between Drosophila HSF
and mammalian HSF1. As was reported for the Fos leucine zipper
homodimer and influenza virus hemagglutinin (65-67), low pH-induced
changes in salt bridges or hydrogen bonds are likely to impair
interactions stabilizing the structure of the HSF monomer and/or
strengthen interactions between subunits of the HSF trimer. In either
case, the association constant would be accordingly increased. Indeed,
an amino acid substitution on the conserved histidine 179 (in the
HR-A/B regions) of mouse HSF1 led to derepression of trimerization
(38). Further mutagenesis and biophysical studies are necessary to
elucidate the mechanism by which HSF is responsive to low pH.
Both analytical ultracentrifugation and gel filtration analyses show at
most about 10-fold decrease in the equilibrium trimer dissociation
constant of Drosophila HSF when buffer pH is decreased from
7.4 to 6.6 at 20 °C. In living cells, heat shock leads to a smaller
change of intracellular pH (from pH 7.36 to pH 7.05), which corresponds
to ~3-fold decrease in the Kd. However, as heat,
oxidation, and low pH show synergistic effects on Drosophila HSF activity in vitro, the effects of high temperature could
be amplified by the moderate intracellular acidification associated with heat stress, and possibly by secondary, oxidative effects in
vivo (68, 69). In addition, the rapid DNA binding of HSF trimers
and the stability of the protein-DNA complex would drive the
equilibrium further toward the trimeric state of HSF.
The failure to observe a lowered intracellular pH for certain heat
shock inducers indicates that changes in pH are not obligatory for
activation of HSF trimerization. Other activating mechanisms have been
proposed, including post-translational modification and the loss of
feedback repression by heat shock proteins (25-37). Our results show
no significant difference in the trimerization constants between
nonshocked and heat-shocked Drosophila HSFs overexpressed in
Sf9 cells. Similar experiments using endogenous HSFs partially
purified from nonshocked and heat-shocked SL2 cells reveal only
2-3-fold differences in the equilibrium trimerization constants for
the two forms of HSF.2 Hence,
heat-induced post-translational modifications may not be essential for
regulating the trimerization and DNA binding activities of HSF. This
conclusion concurs with previous studies showing that trimer formation
and DNA binding of HSF1 are independent of inducible phosphorylation in
mammalian cells (31, 36); similar findings were obtained in a recent
study of Drosophila HSF.3 Some effects of
phosphorylation on the transcriptional competence of HSF have been
reported (32-34, 36). However, there is increasing evidence showing
that heat shock proteins are involved in regulation of HSF
transactivation and recovery after heat shock (30, 48, 70). Reversible
effects of heat shock proteins on HSF trimerization, DNA binding, or
transcriptional activity have not yet been demonstrated with a purified
system. It will be of interest to analyze the direct influence of
environmental conditions and heat shock proteins on HSF activity using
the in vitro system we have developed.
INTRODUCTION
Top
Abstract
Introduction
References
-helical coiled-coil structure (12-16).
The domain responsible for transactivation is generally located in the
carboxyl-terminal region of HSF and shows less sequence conservation
between organisms when compared with the DNA-binding and trimerization
domains (17). The activities of trimerization and transactivation
domains are subject to control by other regions of HSF (18-24).
EXPERIMENTAL PROCEDURES
1 cm
1 at 276 nm,
was calculated according to Pace et al. (45).
) + E; dimer only,
A230,r = Ab·exp(2
) + E; trimer only,
A230,r =
Ab·exp(3
) + E; monomer
trimer equilibrium, A230,r =
Ab·exp(
) + 3Ab3/
2302·exp(3
) + lnKa + E, where
= M(1
)
2(r2
rb2)/2RT,
and Ka is the trimer equilibrium association
constant.
is absorbance at 230 nm at the radial position
r, and Ab is absorbance at the cell
bottom. M refers the molecular mass of the monomer protein,
76,933 daltons, calculated from the predicted amino acid sequence and
confirmed by matrix-assisted laser desorption ionization-time of flight
mass spectrometry with an error of ± 200 daltons; E is
the base line;
230 is the molar extinction coefficient
of the monomer at 230 nm.
is the partial specific volume,
0.723 ml/g, calculated according to Perkins (46);
is
solvent density (47);
is the rotor angular velocity;
R is the universal gas constant, and T is
absolute temperature. The trimer dissociation constant
Kd equals to 1/Ka.
-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa)
(Bio-Rad) were used in parallel as standards. The elution profiles at
215 nm were recorded. The trimer equilibrium dissociation constant is
defined as Kd = [M]3/[T], where for a known
polypeptide concentration C, the monomer concentration is
[M] = C × [area]M/([area]M + [area]T), and the
trimer concentration is [T] = (C
[M])/3.
RESULTS
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Fig. 1.
pH dependence of HSF monomer-trimer
equilibrium. A, percent trimer plotted as a function of
pH. 0.5 µM HSF (NS) (empty squares) and HSF
(HS) (filled squares) were allowed to equilibrate (20 °C
overnight) at the indicated pH in 25 mM sodium phosphate
buffer (for pH values 5.6-7.4) or Tris-HCl (for pH values 7.4-8.3)
containing 150 mM KCl, 1 mM MgCl2,
0.2 mM DTT, and 5% glycerol and analyzed by Superose 6 gel
filtration chromatography. The percent trimer was derived from the
integrated areas under the absorbance peaks corresponding to monomer
and trimer. B, gel filtration profiles showing reversible
induction of HSF trimerization by low pH. 0.5 µM HSF (NS)
samples were equilibrated (20 °C overnight) in 20 mM
sodium phosphate, 150 mM KCl, 1 mM
MgCl2, 0.2 mM DTT, and 5% glycerol at pH 7.4 and 6.6, respectively. An aliquot at pH 6.6 was dialyzed against pH 7.4 buffer and allowed to re-equilibrate for 6 h at 20 °C. At each
step, equivalent amounts of HSF protein were analyzed by Superose 6 chromatography. A small non-native-like peak on the shoulder of the
monomer (~5% of total HSF) can be observed upon incubation at low
pH. This species could not be converted to the monomer or trimer form
and was excluded from the calculations.
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Fig. 2.
Equilibrium analysis of HSF by analytical
ultracentrifugation. Lower panel, typical distribution
of the absorbencies of HSF versus radial position. Data
(open circles) were fitted to a monomer (-·-· -),
dimer (---), trimer (· · ·) and monomer-trimer equilibrium
(---). Upper panel, distribution of the residuals
(open circles) for the best-fit monomer-trimer equilibrium.
The typical weighted root mean square error for monomer-trimer
equilibrium is ~8 × 10 3 optical density. This
measurement was performed with 1 µM HSF (NS) polypeptide
in a buffer containing 20 mM sodium phosphate (pH 7.4), 150 mM KCl, 1 mM MgCl2, 0.2 mM DTT and 5% glycerol at 12,000 rpm and 20 °C. Similar
results were obtained for HSF (HS). Inset: 8%
SDS-polyacrylamide gel electrophoresis and Western blotting showing
integrity of HSF before and after analytical ultracentrifugation for 3 days at 20 °C.
Comparison of equilibrium dissociation constants of HSFs determined by
analytical ultracentrifugation and gel filtration chromatography
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Fig. 3.
Low pH activates DNA binding of HSF. 0.2 µM purified HSFs were equilibrated at the indicated pH in
25 mM sodium phosphate buffer (pH 6.2-7.4) containing 150 mM KCl, 1 mM MgCl2, 0.2 mM DTT, and 5% glycerol at 20 °C overnight and analyzed
by gel mobility shift assay and Western blotting. Heat-shocked samples
(pH 7.4, 36 °C/20 min) were run in parallel for comparison.
Synergistic effects of heat, oxidation, and low pH on the equilibrium
dissociation constants of Drosophila HSF
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Fig. 4.
Heat shock induces intracellular
acidification in Drosophila SL2 cells.
Intracellular pHi was measured using the fluorescent pH
indicator BCECF. A, calibration curve obtained by plotting
the average fluorescence ratio at each pH value. The measurements were
performed at either 23 °C (open circles) or 37 °C
(filled squares). The line was fitted by polynomial
regression of degree three, which was used to convert the measured
fluorescence ratios into pHi values. B, plot of the
intracellular pHi as a function of time during heat shock and
recovery. Under normal conditions, the resting intracellular
pHi of SL2 cells is 7.36 ± 0.02 (n = 22)
at 23 °C. After heat shock for 10 min at 37 °C, the pHi
drops to 7.05 ± 0.02 (n = 7).
Effects of heat shock inducers on the intracellular pHi of
Drosophila SL2 cells
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are grateful to J. Wisniewski for constructing the wild-type Drosophila HSF baculovirus expression vector. We thank members of our laboratory for helpful discussion and comments on the manuscript.
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FOOTNOTES |
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* This work was supported by the Intramural Research Program of the National Cancer Institute (to C. W.) and the Postdoctoral Fellowship from the American Cancer Society (to M. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Mokpo National University, Dept. of Chemistry,
61, Dolim-Ri, Chonggye-Myon, Muan-Gun, Chonnam, Korea, 534-729.
§ To whom correspondence should be addressed: NIH Bldg. 37, Rm. 5E26, Bethesda, MD 20892. Tel.: 301-496-3029; Fax: 301-435-3697; E-mail: carlwu{at}helix.nih.gov.
The abbreviations used are: HSF, heat shock factor; HSE, heat shock element; HSP, heat shock protein; NS, nonshocked; HS, heat-shocked; DTT, dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; BCECF, 2',7'-bis-(2- carboxyethyl)-5,6-carboxyfluorescein.
2 M. Zhong and C. Wu, unpublished observations.
3 Fritsch, M., and Wu, C. (1999) Cell Stress Chaperones, in press.
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REFERENCES |
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