From the
hsp70 proteins of both eukaryotes and prokaryotes possess both
ATPase and peptide binding activities. These two activities are crucial
for the chaperone activity of hsp70 proteins. The activity of DnaK, the
primary hsp70 of Escherichia coli, is modulated by the GrpE
and DnaJ proteins. In the yeast Saccharomyces cerevisiae, the
predominant cytosolic hsp70, Ssa1p, interacts with a DnaJ homologue,
Ydj1p. In order to better understand the function of the Ssa1p/Ydj1p
chaperone, the effects of polypeptide substrates and Ydj1p on Ssa1p
ATPase activity were assessed using a combination of steady-state
kinetic analysis and single turnover substrate hydrolysis experiments.
Polypeptide substrates and Ydj1p both serve to stimulate ATPase
activity of Ssa1p. The two types of effector are biochemically
distinct, each conferring a characteristic K
All cells respond to certain environmental stresses by inducing
the expression of a stereotypical set of proteins known as heat shock
proteins
(1) . The most evolutionarily conserved of these
proteins is known generically as hsp70. hsp70 is a multifunctional
protein with a highly conserved N-terminal ATPase domain of 44 kDa
followed by a somewhat less conserved peptide binding domain of 18 kDa
and a variable domain at its extreme C terminus
(2, 3, 4) . Genetic and biochemical experiments
in Escherichia coli established the existence of two
``cohort'' proteins which modulate the activity of hsp70 in
that organism: DnaJ and GrpE
(5) . Numerous homologues of DnaJ
and GrpE have since been found in other organisms. All DnaJ homologues
are defined by the presence of a conserved motif homologous to the
N-terminal domain of DnaJ in E. coli, but as a group, they are
considerably more heterogeneous in both size and primary sequence than
are hsp70s
(6) . More divergent still are GrpE homologues, which
have only been identified in prokaryotes
(7) and mitochondria
(8) to date.
Through the use of the genetic techniques
available for the study of Saccharomyces cerevisiae, at least
10 hsp70 genes have been identified and studied in this simple
eukaryote. Of these, six encode confirmed cytosolic proteins, products
of two genetic subfamilies: SSA1-4 and SSB1-2(4) . The SSA subfamily is essential. At least one
of the SSA gene products must be present in significant
quantity to ensure viability. Loss of SSA function has
pleiotropic effects, including perturbations in heat shock regulation
and defects in protein localization
(9, 10) . The
SSB subfamily consists of two highly homologous genes whose
products are part of the translation apparatus and appear to facilitate
protein synthesis
(11) . The SSE subfamily ( SSE1 is identical to the recently isolated MSI3 gene) is a
recently identified set of two closely related genes whose products
appear to be involved in calmodulin- and cAMP-dependent signaling
pathways
(12, 13) and may represent another cytosolic
hsp70 subfamily. There is every reason to expect that similarly
extensive hsp70 families also exist in other eukaryotes.
Recent work
in several laboratories has established hsp70 as a molecular chaperone,
a member of an expanding family of proteins whose function is to assist
in the proper folding of polypeptides. The most complete understanding
of hsp70 function has been achieved in studies of E. coli,
whose principal hsp70 (encoded by the dnaK gene) has been
shown to interact with both DnaJ and GrpE. Although some functions,
It is not clear to what degree conclusions
drawn from the analysis of the hsp70 system of E. coli can be
extended to eukaryotic hsp70s and their cohort proteins. In order to
better understand the function of hsp70 in the eukaryotic cytosol, we
undertook a biochemical study of Ssa1p, the major heat-inducible
species in S. cerevisiae. We compared the effects of Ydj1p and
polypeptide substrates on the ATPase activity of Ssa1p through a
combination of steady-state kinetic approaches and single turnover
substrate hydrolysis experiments. The focus of these experiments was to
determine the effect of polypeptide substrates and Ydj1p on the
regulation of Ssa1p activity by ATP and ADP.
Since
the stimulation of Ssa1p ATPase activity by either peptide or Ydj1p
changes its enzymatic properties depending upon the K
It is clear from the work presented here that K
The distinct
effects of Ydj1p and peptide substrates on Ssa1p ATPase activity were
evident in studies of the K
ATPase assays containing both
peptide and Ydj1p at several KCl concentrations further showed that
peptide and Ydj1p interact differently with Ssa1p. At 5 mM
KCl, a concentration which prevented peptide stimulation of ATPase
activity, the presence of peptide was able to interfere with Ydj1p
dependent stimulation. This result by itself could lead one to conclude
that peptide and Ydj1p compete for the same binding site. However, when
both effectors were present in a reaction containing 80 mM
KCl, a concentration which allowed modest stimulation of ATPase by
either Ydj1p or peptide A7 (4.3- and 3.7-fold, respectively), the
resulting 7-fold increase in activity was greater than that obtained
with either effector alone. The combinatorial effect of Ydj1p and
peptide A7 suggests that they interact with distinct sites on Ssa1p or
that binding of the two effectors is separated temporally. Since these
experiments were done at near saturating levels of both Ydj1p and
peptide A7, the latter possibility would require that either Ydj1p or
peptide act transiently to change the biochemical characteristics of
Ssa1p. The available data do not allow us to distinguish between these
models at present.
Although Ydj1p and peptides have distinct effects
on Ssa1p ATPase activity, the two effectors share both the capacity to
accelerate the hydrolysis of bound ATP and to destabilize the binding
of ATP to Ssa1p. Studies of the fate of a
[
Ydj1p accelerated the hydrolysis of bound ATP by as much as 20-fold
at 150 mM KCl. This acceleration is in contrast to the
steady-state ATPase activity, which is virtually unaffected under the
same conditions, suggesting that ADP release (Fig. 7,
k
The DnaK/DnaJ/GrpE system of E. coli is often cited as a paradigm for hsp70 function. Although the
study of the E. coli system has been very informative, our
results suggest that there may be important differences between the
hsp70s of prokaryotes and those of the eukaryotic cytosol. Upon
incubation with [
Because the ATP
Despite the differences between Ssa1p and DnaK, the results
presented above are generally in good agreement with recent models of
hsp70 function. It has been suggested that hsp70 is regulated by
occupancy of the ATPase domain by either ATP, which promotes rapid but
unstable binding of polypeptide substrates, or ADP, which favors stable
binding of polypeptide substrates
(38, 39) . Indeed,
recent results from other laboratories suggest that ATP hydrolysis is
not stoichiometrically coupled to cycles of peptide binding and release
(25, 38) . In such a model, GrpE would serve to convert
hsp70 from an ADP- to an ATP-bound form through the exchange of bound
ADP for ATP. DnaJ would have the opposite effect, favoring an ADP-bound
form through the hydrolysis of bound ATP to ADP
(40) . Our
results suggest that the requirement for modulators of hsp70 activity
may be determined by the enzymatic properties of that particular hsp70
protein. In E. coli, DnaJ appears to interact with certain
polypeptide substrates prior to DnaK, possibly to target these
substrates for DnaK action
(16, 26, 41, 42, 43) .
Preliminary evidence indicates that Ydj1p prevents the aggregation of
unfolded rhodanese,
The data were analyzed using a
nonlinear regression routine as described under ``Materials and
Methods.'' Units are moles of ATP hydrolyzed per mole of Ssalp min
( V
Reaction mixtures were
prepared and reactions carried out at 0 °C (on ice) or 30 °C as
described in the legend to Fig. 3. Half-life ( t) in minutes
was calculated using the formula t = 0.301
We thank Paul Bertics for helpful discussions and
critical reading of the manuscript. We also thank Jill Johnson, Shikha
Laloraya, Ed Maryon, and Eva Ziegelhoffer for helpful comments on the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
dependence on Ssa1p ATPase activity. However, in single turnover
ATP hydrolysis experiments, both polypeptide substrates and Ydj1p
destabilized the ATP
Ssa1p complex through a combination of
accelerated hydrolysis of bound ATP and accelerated release of ATP from
Ssa1p. The acceleration of ATP release by Ydj1p is a previously
unidentified function of a DnaJ homologue. In the case of
Ydj1p-stimulated Ssa1p, steady-state ATPase activity is increased less
than 2-fold at physiological K
concentrations, despite
a 15-fold increase in the hydrolysis of bound ATP. The primary effect
of Ydj1p appears to be to disfavor an ATP form of Ssa1p. On the other
hand, peptide stimulation of Ssa1p ATPase activity was enhanced at
physiological K
concentrations, supporting the idea
that cycles of ATP hydrolysis play an important role in the interaction
of hsp70 with polypeptide substrates. The enhanced ATP dissociation
caused by both polypeptide substrates and Ydj1p may play a role in the
regulation of Ssa1p chaperone activity by altering the relative
abundance of ATP- and ADP-bound forms.
DNA replication for example, require all three proteins
(14, 15) , others such as bacteriophage P1 replication
require only DnaK and DnaJ
(16) . The ATPase and peptide binding
activities of hsp70 have been studied by several groups. In general,
hsp70s possess an intrinsic ATPase activity of 0.01-0.03 (mol of
ATP hydrolyzed/mol of hsp70/min) which, in many cases, is stimulated by
the presence of certain peptides or unfolded proteins
(17) .
Peptide substrates appear to be bound in an extended conformation
(18) and are usually enriched for certain hydrophobic groups
(19, 20, 21) . Peptide binding and release is
controlled by the ATPase domain
(22, 23) . The structure
of the ATPase domain has been solved to 2.4 angstrom resolution,
revealing a striking similarity to actin and the nucleotide binding
sites of hexokinase and glycerol kinase
(2, 24) . The
presence of ATP in the nucleotide binding site generally disfavors
stable binding of peptide or unfolded protein substrates, whereas ADP
tends to stabilize such interactions. This effect has been shown to be
K
-dependent
(25) . Stimulation of ATPase
activity has also been shown for DnaJ and GrpE, which can act
synergistically in vitro to increase ATPase activity by up to
50-fold. DnaJ acts primarily to increase the rate of ATP hydrolysis
while GrpE increases the rate of ATP and ADP release
(5) .
Although no cytosolic GrpE homologue has been identified in yeast,
Ydj1p, a cytosolic DnaJ homologue, has been shown to interact
genetically with Ssa1p, the most abundant cytosolic hsp70 in
yeast.
(
)
Furthermore, purified Ydj1p has been
shown to stimulate the ATPase activity of Ssa1p in vitro.
Unlike DnaJ, which confers only 2-fold stimulation on DnaK ATPase
activity, Ydj1p can stimulate Ssa1p ATPase activity more than 10-fold
(27, 28) .
Proteins and Peptides
Peptides A5 (APRLRFTSL)
and A7 (RRLIEDAETAARG) were obtained from Sigma (catalog numbers A5308
and A7433, respectively) and used as 5 mg/ml stock solutions. Both
peptides were devoid of measurable ATPase activity. Reduced
carboxymethylated -lactalbumin (CMLA,
(
)
prepared as a 10 mg/ml stock solution) was also obtained
from Sigma. This preparation was boiled to remove contaminating ATPase
activity. Acetylated bovine serum albumin (BSA) was obtained from New
England Biolabs. Protein concentrations were measured using the
Bradford assay (Bio-Rad protein assay) and ovalbumin (Sigma) as a
standard. Ssa1p was purified essentially as described previously
(27) from a strain containing deletions in SSA2,
SSB1, and SSB2 (kindly provided by B. Diane Gambill,
this laboratory) and was stored at -70 °C at a concentration
of
8 µM in a buffer containing 20 mM Tris
HCl (pH 7.5), 20 mM NaCl, 5 mM MgCl
, 5
mM 2-mercaptoethanol, and 10% (v/v) glycerol. DnaK was
purified essentially as reported by others
(5) . For Ydj1p
purification, a T7 expression construct in E. coli, analogous
to that described by Caplan et al.(29) , was
constructed using a genomic YDJ1 clone (kindly provided by Wei
Yan, this laboratory). Purification of recombinant Ydj1p was as
described previously
(27) , yielding a preparation of >95%
purity. The final concentration of Ydj1p was 130 µM in a
buffer containing 20 mM Tris HCl (pH 7.5), 20 mM
NaCl, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol.
ATPase Assays
Assays were carried out at 33 °C
in buffer containing 10 µM ATP (1 µCi
[-
P]ATP per reaction), 50 mM
Tris-HCl (pH 7.5), 50 mM KCl, 2 mM MgCl
,
and 5 mM 2-mercaptoethanol unless otherwise specified. The
final concentration of Ssa1p was between 0.18 and 0.33 µM,
as noted in the figure legends. 40-µl reaction mixtures were
pre-equilibrated for 10 min at the assay temperature prior to addition
of substrate to initiate the reaction. At several time points, aliquots
were removed and applied to polyethyleneimine TLC plates and developed
essentially as published elsewhere
(5) . Either liquid
scintillation counting or PhosphorImager analysis (Molecular Dynamics,
Inc.) was used to quantitate the fraction of ATP hydrolyzed to ADP at
each time point. Slopes were determined by linear regression analysis
(Cricketgraph software). For the K
determinations, assays were carried out in the presence of 200
mM KCl and 0 µM ADP (0.125, 0.25, 0.5, 1.0, and
2.5 µM ATP), 15 µM ADP (0.5, 1.0, 3.0, 5.0,
and 10 µM ATP), and 40 µM ADP (1.0, 3.0, 5.0,
10, and 17.5 µM ATP). Ssa1p was present at 0.34
µM and 0.07 µM, depending on whether it was
assayed alone or in the presence of effector (peptide or Ydj1p),
respectively. Peptide A7 and Ydj1p were present at 145 and 0.8
µM, respectively. The data were analyzed using a nonlinear
regression routine
(30) . [
-
P]ATP
Ssa1p Complex
Formation-In a slight modification of the procedure used
previously in the study of DnaK
(5) , 60 µg of Ssa1p was
incubated for 5 min at 21 °C with 100 µCi of
[
-
P]ATP in a 100-µl reaction containing
200 mM KCl, 10 mM MgCl
, 20
µM ATP, and 10% (v/v) glycerol. The reaction was chilled
on ice and the [
-
P]ATP
Ssa1p complex
was purified away from free ATP by fractionation over a 8
0.5-cm Sephadex G50 column equilibrated with standard buffer (150 or
200 mM KCl, 25 mM Tris-HCl (pH 7.5), 2.5 mM
MgCl
, and 5 mM 2-mercaptoethanol) at a flow rate
of about 0.2 ml/min. Fractions containing the protein peak (as
determined by amido black staining of column fractions spotted onto
nitrocellulose) were pooled and frozen on dry ice prior to storage at
-70 °C. Two such preparations were used in this study:
preparation 1 is 0.8 µM Ssa1p, 150 mM KCl and 2.3
nCi/µl and was used in the experiments shown in Figs. 4 and 6 and
, preparation 2 is 3 µM Ssa1p, 200
mM KCl, and 6.8 nCi/µl and was used in the experiment
shown in Fig. 5. In assays of the hydrolysis of bound ATP, 8
µl of complex was added to a pre-equilibrated microcentrifuge tube
containing 2 µl of additional reaction components. 2-µl
aliquots were removed at the indicated time points and added to a tube
containing 1 µl of 4 M formic acid to terminate the
reaction. Samples were then applied to TLC plates as described for
ATPase assays. Isolation of the
-
P-labeled
nucleotide
DnaK complex was as described by Liberek et al.(5) , yielding a preparation which was 8.3 µM
DnaK and 2.5 nCi/µl.
Figure 5:
Quenching of bound ATP hydrolysis by
excess unlabeled ADP in the presence and absence of CMLA and Ydj1p. The
rate of hydrolysis of bound ATP was measured at 30 °C as in Fig. 3
using purified [-
P]ATP
Ssa1p to which
was added either BSA ( A), CMLA ( B), or Ydj1p
( C). Duplicate reaction tubes were prepared, one of which
contained a large molar excess of unlabeled ADP (100 µM
final concentration, filled triangles), the other contained no
added nucleotide ( open squares). The final concentrations of
the other components were as described in the legend to Fig. 3. The
data for hydrolysis in the absence of ADP are taken from Table II,
experiment 1.
Intrinsic Ssa1p ATPase Activity Is
K
As a first step in characterizing
the effects of peptide and Ydj1p on Ssa1p ATPase activity, we extended
the characterization of the intrinsic ATPase activity of Ssa1p. The
K-dependent
dependence of Ssa1p ATPase activity was measured as
a function of K
concentration and ionic strength. The
intrinsic Ssa1p ATPase activity was K
-dependent, with
maximum activity occurring at about 10 mM KCl
(Fig. 1 A). Activity dropped approximately 8-fold as the
KCl concentration was lowered to below 1 mM. This may be an
underestimate of the K
dependence, since, in the
absence of added KCl, Ssa1p ATPase activity is near the limit of
detection in these assays. At KCl concentrations above 20 mM,
activity was reduced slightly. This dependence is clearly not simply an
ionic strength effect, as the presence of compensating levels of NaCl
does not abolish the K
dependence ( closed
circles). This experiment also shows that the concentration of the
counterion, Cl
, has no appreciable effect on Ssa1p
ATPase activity. Since full activity of Ssa1p ATPase requires
K
, it is likely that the enzyme has one or more
K
binding sites.
Figure 1:
K dependence of
intrinsic and stimulated Ssa1p ATPase. ATPase activity was measured as
a function of KCl concentration either alone ( A) or in the
presence of 150 µM peptide A7 ( B) or 0.50
µM Ydj1p ( C). The concentration of Ssa1p was 0.30
µM ( A and B) or 0.18 µM
( C). Assays were done at the indicated concentrations of KCl
either in the absence ( open squares) or presence of
compensating concentrations of NaCl such that the total monovalent
cation concentration was 300 mM ( closed circles).
Note that the y axis differs substantially between A,
B, and C. Similar experiments showed analogous KCl
dependencies for intrinsic and peptide- or Ydj1p-stimulated
activity.
The K dependence
of Ssa1p ATPase activity is due in part to the dependence of the
K
for ATP on K
concentration. The dependence of intrinsic ATPase activity on
substrate concentration was measured at three different KCl
concentrations and the data were analyzed using a nonlinear regression
routine
(30) . A dramatic decrease in the
K
for ATP from 4.2 µM at 2.5
mM KCl to 0.19 µM at 200 mM KCl was
observed. V
also increased significantly as KCl
concentration was increased from 2.5 to 15 mM ().
The K
values suggest that binding of
substrate may be facilitated by the presence of K
ions. The increase in
V
/ K
with
increasing KCl concentration suggests that the efficiency of catalysis
is highest at physiological (>100 mM) K
concentrations.
Distinct Interactions of Peptides and Ydj1p with
Ssa1p
Since Ssa1p is K-dependent, we asked if
either peptide (Fig. 1 B) or Ydj1p
(Fig. 1 C) affect this dependence. In addition to their
ability to stimulate ATPase activity (note differences in the y axes of the graphs), peptide A7 and Ydj1p also altered the salt
dependence of Ssa1p ATPase activity. The presence of peptide had two
effects: 1) optimum activity was shifted to higher K
concentrations (50-100 mM KCl), and 2) ATPase
activity was inhibited somewhat at concentrations of K
below approximately 20 mM (Fig. 1 B). An
unrelated stimulatory peptide, A5, had the same effect on Ssa1p ATPase
activity, suggesting that this is a general response of Ssa1p to short
peptide substrates (data not shown). Reduced CMLA, previously shown by
others to be a polypeptide substrate for Ssa1p
(27) , resulted
in a similar shift in the K
dependence, but did not
exhibit the ATPase inhibition at low K
concentrations
seen with short peptides (data not shown). Unlike peptide, Ydj1p did
not alter the concentration of K
at which maximum
activity was achieved. However, although the activity of both intrinsic
and peptide-stimulated ATPase remained relatively high at physiological
K
concentrations, the Ydj1p-stimulated activity was
greatly reduced at KCl concentrations above 100 mM, such that
less than a 2-fold stimulation of ATPase activity was observed at 200
mM KCl. This inability of Ydj1p to stimulate ATPase activity
did not depend specifically on K
, as high
concentrations of NaCl were able to substantially reduce stimulation
even at ``optimal'' K
concentrations
(Fig. 1 C, filled circles). These data clearly show that
while Ydj1p is capable of up to a 40-fold stimulation of ATPase
activity at 10-20 mM K
, at
physiological K
concentrations, only modest
stimulation of Ssa1p ATPase activity can be achieved by Ydj1p.
concentration and the effector, the combined effect of peptide
and Ydj1p was examined at several KCl concentrations in the hope that
this would allow us to distinguish between the mechanisms of action of
the two effectors. Concentrations of both Ydj1p and peptide A7 were
chosen so as to give near maximal stimulation. KCl concentrations were
chosen so as to separate the activities of peptides and Ydj1p, based on
the results presented above. At 5 mM KCl, ATPase activity of
Ssa1p increased nearly 20-fold in the presence of a 5-fold molar excess
of Ydj1p. The addition of peptide A7 by itself had no effect on ATPase
activity but strongly inhibited the stimulatory effect of Ydj1p,
resulting in a 6-fold reduction in the Ydj1p-stimulated activity
(Fig. 2 A). When similar assays were done at 50
mM KCl, intrinsic ATPase activity was about 3-fold higher than
that seen at 5 mM KCl (Fig. 2 B). Ydj1p or
peptide A7 further stimulated this activity by about 5- and 3-fold,
respectively. In the presence of both effectors, activity was at a
level comparable with that observed with Ydj1p alone. A further
increase in the KCl concentration to 80 mM did not
substantially alter the intrinsic ATPase activity
(Fig. 2 C). Stimulation of ATPase activity by Ydj1p at 80
mM KCl was 4.3-fold, whereas peptide A7 stimulation was
3.7-fold. The presence of both effectors resulted in a 7-fold increase
in ATPase activity, significantly greater than the stimulation observed
with either Ydj1p or peptide alone, suggesting that under certain
conditions, the effects of peptides and Ydj1p are cumulative. Analogous
experiments in which peptide A5 and Ydj1p were combined also resulted
in greater stimulation of Ssa1p ATPase than either effector by itself
(data not shown), suggesting that the combinatorial stimulation seen at
higher KCl concentrations does not depend on the identity of the
stimulatory peptide.
Figure 2:
Combined effects of peptide A7 and Ydj1p
on Ssa1p ATPase at various KCl concentrations. ATPase activity was
assayed under standard conditions with three different KCl
concentrations, 5 mM ( A), 50 mM
( B), and 80 mM ( C). Ssa1p was 0.33
µM in all assays. Reactions contained the indicated
concentrations of Ydj1p or peptide. In those reactions containing both
effectors, the highest concentration (1600 nM Ydj1p and 200
µM peptide A7) was used. Assays were done in triplicate.
Activity is expressed as moles of ATP hydrolyzed per mole of Ssa1p per
min. Error bars denote the standard deviation for each
activity determination.
Both Polypeptide Substrates and Ydj1p Accelerate the
Hydrolysis of Bound ATP by Ssa1p
To further assess the roles of
peptides and Ydj1p in modulating Ssa1p ATPase activity, their effects
on the hydrolysis and stability of bound ATP were investigated. As
described under ``Materials and Methods,'' a
[-
P]ATP
Ssa1p complex was allowed to
form and then purified away from free ATP by size exclusion
chromatography. Peptide A5 or A7, CMLA, acetylated BSA, or Ydj1p was
added to the isolated complex, and the rate of hydrolysis of bound ATP
was measured. BSA was used as a control for nonspecific protein effects
and was found to have the same effect as buffer alone (data not shown).
The ATP complex decayed to an ADP complex exponentially, characteristic
of a first order reaction. Semi-log plots of the data yielded slopes
which were used to determine the rate of hydrolysis of bound ATP in the
presence of each effector. The addition of either peptide A5 or A7,
CMLA, or Ydj1p to the ATP
Ssa1p complex resulted in a substantial
increase in the rate of hydrolysis of bound ATP at 30 °C
(Fig. 3, ). Ydj1p had the most dramatic effect on
the hydrolysis of bound ATP, decreasing the half-life of the
Ssa1p
ATP complex by up to 20-fold. Polypeptide substrates also
accelerated the hydrolysis of bound ATP substantially, with t values approximately 3-fold lower than in the absence of
effectors. It should be noted that the rate constants obtained for the
hydrolysis of bound ATP in the absence of effectors are reasonably
close to the value of k
(4.5
10
min
) derived from the data in
, indicating that hydrolysis of bound ATP is probably the
rate-limiting step in the ATPase reaction. Because of the very rapid
hydrolysis of bound ATP by Ssa1p in the presence of Ydj1p, a set of
analogous experiments was performed at 0 °C, the objective of which
was to obtain more accurate values for the half-life of the complex in
the presence and absence of effectors. While the hydrolysis of bound
ATP by Ssa1p or CMLA-stimulated Ssa1p was approximately 30 times slower
at 0 °C, the hydrolysis of bound ATP by Ydj1p-stimulated Ssa1p was
reduced by less than 5-fold under the same conditions (),
further suggesting that polypeptide substrates and Ydj1p differ
mechanistically in their effects on Ssa1p.
Figure 3:
Effect of peptide A7, A5, CMLA, and Ydj1p
on the hydrolysis of bound ATP.
[-
P]ATP
Ssa1p was combined with either
BSA (1 mg/ml final concentration), peptide A5 (316 µM
final concentration), peptide A7 (289 µM final
concentration), CMLA (73 µM final concentration), or Ydj1p
(4.3 µM final concentration) and incubated at 31 °C.
Aliquots were removed at the indicated times and quantitated for ATP
hydrolysis as described under ``Materials and Methods.'' The
final concentration of Ssa1p was 0.64
µM.
Polypeptide Substrates and Ydj1p Increase the Rate of
Dissociation of ATP from an ATP
To assess the
stability of the nucleotideSsa1p Complex
Ssa1p complex, reactions were allowed
to proceed to about 50% ATP hydrolysis and then rapidly subjected to
another round of chromatography to separate free nucleotide from the
remaining complex. In the control reaction, more than 95% of the
nucleotide in the remaining complex was ATP, whereas most of the free
nucleotide was ADP, suggesting that ATP was stably bound and that ADP
release was complete within minutes of ATP hydrolysis (Fig. 5,
open squares). However, in the presence of peptide A7, ADP
represented about 40% of the bound nucleotide. Of the free nucleotide,
40% was ATP, indicating that the stability of the ATP
Ssa1p
complex was reduced in the presence of peptide A7 (Fig. 4,
filled squares). The stability of the ATP
Ssa1p complex
in the absence of peptide is particularly striking when one considers
that the control reaction was incubated at 30 °C 12 times longer
than the A7-stimulated reaction to achieve a similar degree of ATP
hydrolysis, thereby allowing more time for the ATP
Ssa1p complex
to dissociate. Given the extremely rapid hydrolysis of bound ATP in the
presence of Ydj1p at both 30 °C and 0 °C, it was not possible
to recover a comparable nucleotide
Ssa1p complex in the presence
of Ydj1p to perform similar experiments.
Figure 4:
Stability of ATPSsa1p complex in the
presence and absence of peptide A7. A 25-µl aliquot of isolated
[
-
P]ATP
Ssa1p complex (3
µM Ssa1p, 170 nCi/aliquot) was incubated at 33 °C for
either 6 min (unstimulated, open squares) or 30 s (peptide
A7-stimulated, 97 µM final concentration, filled
squares) to achieve approximately 50% ATP hydrolysis. The tubes
were then placed on ice and 5 µl of a 50% (v/v) glycerol solution
was added. The samples were applied to a 8
0.5-cm Sephadex G50
column equilibrated with standard buffer (see ``Materials and
Methods'') and four-drop fractions (approximately 120 µl) were
collected. The quantity of
P in each fraction was
determined by liquid scintillation counting. Aliquots of fractions 6
and 16 (corresponding to bound and free nucleotide, respectively) were
applied to polyethyleneimine TLC plates and processed as described
under ``Materials and Methods'' in order to determine the
ratio of ATP to ADP present.
In order to further
investigate the effects of polypeptide substrates and Ydj1p on
[-
P]ATP
Ssa1p complex stability, the
ability of excess unlabeled ADP to quench the hydrolysis of bound
[
-
P]ATP was determined. The quenching of
[
-
P]ATP hydrolysis by ADP depends on the
ability of ADP to competitively inhibit ATP hydrolysis under
steady-state conditions (as described below). Although the presence of
100-fold excess unlabeled ADP should have no effect on the hydrolysis
of bound ATP, [
-
P]ATP, which dissociates
from Ssa1p, will remain unhydrolyzed, because it must compete with the
large pool of free ADP for binding to Ssa1p. When excess unlabeled ADP
(Fig. 5 A, triangles) was added to the
[
-
P]ATP
Ssa1p reaction mixture,
hydrolysis of ATP to ADP no longer followed first order kinetics. The
extent to which ATP hydrolysis was inhibited provided a measure of the
dissociation of the ATP
Ssa1p complex, a process which is also
expected to occur exponentially. An approximation of the rate of ATP
dissociation was obtained by plotting 1 - fraction ATP
hydrolyzed
- fraction ATP hydrolyzed
as a function of time on a semi-log plot and determining the
slope of the line describing the data points. In two separate
experiments, the apparent half-life of the complex was 32 and 48 min
with respect to ATP dissociation. The addition of excess unlabeled ADP
to reactions containing Ydj1p or CMLA resulted in complete quenching of
[
-
P]ATP hydrolysis within several minutes
(Fig. 5, B and C), leading us to conclude that
both CMLA and Ydj1p accelerate the rate of ATP dissociation from Ssa1p
by at least an order of magnitude. A similar degree of quenching
occurred in the presence of peptides A5 and A7 (data not shown),
indicating that destabilization of ATP binding is a general property of
polypeptide substrates of Ssa1p. When unlabeled ATP was substituted for
ADP in analogous quenching experiments, similar results were obtained
(data not shown), indicating that the observed effects of peptides and
Ydj1p do not depend on the identity of the unlabeled nucleotide.
The ATPase Domains of Ssa1p and DnaK Respond Differently
to the Binding of CMLA
Single turnover experiments carried out
with DnaK have clearly shown that GrpE can function to dissociate
nucleotideDnaK complexes
(5) . However, no such activity
has been attributed to polypeptide substrates of DnaK. In order to test
this possibility, we determined the effect of CMLA, which has
previously been shown to form an ATP-sensitive complex with DnaK
(25) , on the stability of an ATP
DnaK complex. While we
were able to isolate a nucleotide
Ssa1p complex with as little as
3% ADP, the purified nucleotide
DnaK complex always contained a
substantial fraction of ADP, varying from 50 to 80% of the total bound
nucleotide in several similar experiments. A similar high percentage of
ADP in the nucleotide
DnaK complex was observed by Liberek et
al.(5) and is probably due to the stability of the
ADP-DnaK complex. Compared with the control reaction (Fig. 6,
squares), a 9-fold stimulation of hydrolysis of bound ATP was
observed in the presence of CMLA ( circles). However, no
quenching of the hydrolysis reaction occurred when a large excess of
unlabeled ADP was present in the reaction ( filled symbols),
clearly indicating that CMLA binding to DnaK did not increase the
ability of ADP to quench ATP hydrolysis and, hence, the rate of ATP
release from DnaK. Thus, a comparison of the effect of CMLA binding on
these two hsp70 proteins (Fig. 5 B versusFig. 6
)
shows that DnaK, unlike Ssa1p, does not release bound ATP appreciably
upon the binding of peptide ligands.
Figure 6:
Effect of CMLA on the stability of an
ATPDnaK complex. The
[
-
P]ATP
DnaK complex was isolated as
described under ``Materials and Methods'' and incubated with
BSA (control, squares) or CMLA ( circles). ADP was
added to 400 µM final concentration in two of the
reactions ( filled symbols). The final concentration of
proteins was 1 mg/ml BSA or 73 µM CMLA and 5
µM DnaK. The addition of ADP to the nucleotide
DnaK
complex in the presence or absence of CMLA reproducibly increased the
rate of ATP hydrolysis about 2-fold.
Both Peptide A7 and Ydj1p Favor an ADP Form of
Ssa1p
Several models for hsp70 function have been proposed. A
prevalent model is based on models for G protein function, in which
modulators of GTPase activity act to favor either the GTP or GDP bound
form of the enzyme
(31) . By analogy, Ssa1p might switch between
two different conformations depending upon whether ATP or ADP is bound
at the ATPase domain. Since both peptides and Ydj1p appear to have
effects on the stability of an ATPSsa1p complex, we asked if
either effector changed the stability of the ADP form of the enzyme.
Because of the instability of the ADP
Ssa1p complex, the effects
of Ydj1p and polypeptide substrates on this complex were difficult to
study directly. However, we found that either peptide or Ydj1p
increased the potency of ADP as an inhibitor of Ssa1p ATPase activity.
The effect of Ydj1p and peptide A7 on the prevalence of an
ADP
Ssa1p complex was measured by determining the inhibition
constant ( K
) for ADP in the presence of either
effector. ATPase assays were performed in the presence of two different
ADP concentrations and in the absence of ADP. In each of these three
experimental conditions, the ATP concentration dependence of the ATPase
activity was determined. Inhibition by ADP was competitive (data not
shown), consistent with a single nucleotide binding site. The intrinsic
ATPase had a K
for ADP of 2.8 ± 0.52
µM under the assay conditions used. In the presence of
peptide A7 or Ydj1p, the K
was reduced to 0.79
± 0.18 µM or 1.2 ± 0.64 µM,
respectively. No significant change in the K
for ATP was observed in the presence of peptide or Ydj1p.
Clearly, both Ydj1p and peptide A7 significantly decrease the
K
for ADP, supporting the conclusion that they
serve to increase the prevalence of an ADP form of Ssa1p.
concentration is important for Ssa1p ATPase activity, since the
ATPase activity of Ssa1p increases nearly 10-fold between 0 and 20
mM KCl. Such a K
dependence is not unique, as
several other ATPases are also known to be K
-dependent
(32) . This dependence may not be characteristic of all hsp70s,
since DnaK
(33) and the yeast mitochondrial hsp70, Ssc1p,
show less than a 2-fold increase in activity as KCl concentration
is increased from 0 to 100 mM. (
)
dependence of this
activity. Ydj1p did not change the K
optimum for Ssa1p
ATPase, although stimulation was much weaker at high ionic strength,
such that approximately 1.5-fold stimulation was observed at
physiological K
concentrations (150-200
mM KCl). In contrast, peptide binding shifted the
[K
] optimum of Ssa1p ATPase to between 50
and 100 mM KCl. Unlike Ydj1p, peptide stimulation of Ssa1p
ATPase persists at physiological K
concentrations. It
should also be noted that maximal stimulation of Ssa1p ATPase by
peptide requires at least a 100-fold higher molar ratio of peptide to
Ssa1p than is required for Ydj1p.
-
P]ATP
Ssa1p complex showed that in
the absence of Ydj1p or polypeptide substrates, the conversion of
ATP
Ssa1p to ADP
Ssa1p is the rate-limiting step in the
reaction, resulting in the accumulation of ATP
Ssa1p
(Fig. 7, k
). Both effectors greatly
stimulated the hydrolysis of bound ATP, by at least 15-fold for Ydj1p
and 3-fold for polypeptide substrates. The addition of polypeptide
substrate or Ydj1p to the complex also resulted in its rapid
dissociation, as evidenced by the ability of excess unlabeled ADP to
quench the ATPase reaction. We estimate that the rate of ATP release
from Ssa1p increased by more than 10-fold in the presence of either
Ydj1p or CMLA. Therefore, polypeptide substrates and Ydj1p destabilize
the ATP
Ssa1p state by increasing both the k
and k
terms (Fig. 7). The
increased ATP dissociation we observed in the presence of CMLA is
similar to the stimulation of nucleotide exchange by unfolded proteins
which has been reported in studies of bovine hsc73
(31) , also
known as hsc70 or bovine brain-uncoating ATPase. However, the
destabilization of nucleotide binding by a DnaJ homologue has not been
reported previously, raising the possibility that this activity might
be a general feature of DnaJ homologues of the eukaryotic cytosol. In a
sense, both peptides and Ydj1p act as ``exchange factors,'' a
role proposed for GrpE in its interaction with DnaK.
Figure 7:
Diagram of the hsp70 ATPase
reaction.
Unlike GrpE,
however, Ydj1p and peptide substrates of Ssa1p also accelerate the rate
of hydrolysis of bound ATP. The degree of stimulation of hydrolysis of
bound ATP by polypeptide substrates is similar to the degree of
stimulation of steady state ATPase activity under similar conditions.
In other words, the change in k is proportional to
the change in steady state ATP turnover ( k
),
indicating that k
probably remains rate-limiting
in the presence of peptide substrates. The similarity of
k
and k
exists in spite of
the increase in the dissociation of ATP from Ssa1p in the presence of
peptides or CMLA. Indeed, our results suggest that, in the presence of
peptide, bound ATP is probably released from the active site more
rapidly than it is hydrolyzed to ADP. Despite this fact, we observe
first order kinetics in the absence of excess unlabeled ADP, suggesting
that the rebinding of ATP (Fig. 7, k
) to the
enzyme is not a rate-limiting step in the reaction. The decreased
K
( e.g. increased affinity) for ADP in
the presence of peptide A7 is consistent with our observation that ATP
release is enhanced by polypeptide substrates and suggests that binding
of peptide substrates would tend to favor an ADP form of Ssa1p.
) from Ssa1p may be rate-limiting in the presence
of Ydj1p. In this respect, Ydj1p appears to function like DnaJ of
E. coli, primarily serving to accelerate the hydrolysis of
bound ATP to ADP. The increased inhibition of ATPase by ADP in the
presence of Ydj1p also suggests that the role of Ydj1p is to favor an
ADP form of Ssa1p. As in the case of peptide-stimulated Ssa1p, the
increased rate of ATP dissociation ( k
),
relative to ADP dissociation ( k
), from
Ydj1p-stimulated Ssa1p should also serve to drive equilibrium toward an
ADP form of the enzyme.
-
P]ATP, both Ssa1p and
DnaK were readily recovered as
P-labeled
nucleotide
hsp70 complexes. In contrast to the DnaK complex, the
P-labeled nucleotide
Ssa1p complex was virtually
devoid of ADP (Fig. 4); indeed, we were unable to isolate an
ADP
Ssa1p complex. However, the nucleotide
DnaK complex
generally contained at least 50% ADP, despite our efforts to minimize
the hydrolysis of ATP during recovery of the complex. Therefore, a
primary difference between the Ssa1p and DnaK ATPase reaction appears
to be the rate of ADP dissociation from the active site
( k
, Fig. 7). Preliminary experiments
indicate that the half-life of the ADP
DnaK complex is
approximately 1-2 min ( k
0.3-0.7
min
) at 23 °C.
(
)
The
apparent instability of ADP
Ssa1p suggests to us that Ssa1p may
not require a GrpE-like nucleotide exchange factor, since the apparent
function of such proteins, by analogy with nucleotide exchange factors
of G proteins
(34) , is to facilitate the release of ADP from
the ATPase domain and permit the binding of ATP. In support of this
idea, preliminary studies indicate that Ssa1p and Ydj1p can together
facilitate the folding of denatured luciferase in
vitro.
(
)
Ssa1p
complex is intrinsically quite stable, one might expect that
stabilization of an ADP form of the enzyme would be an important
function of some modulators of Ssa1p activity. As discussed above, this
appears to be a function of both polypeptide substrates and Ydj1p. In
contrast, the release of ATP from DnaK was not affected by the presence
of CMLA. In the case of Ssa1p, the ATP release activity of polypeptide
substrates and Ydj1p may be required to compensate for the relative
instability of the ADP form of the enzyme. Another function of the ATP
release activity could be to couple chaperone activity to the cellular
ADP:ATP ratio, which reflects the physiologically important
``energy charge''
(35) of the cell. This ratio is
reported to vary between about 0.4 and 2 in S. cerevisiae(36) , depending upon whether growth occurs under anaerobic
or aerobic conditions, respectively. Furthermore, some evidence
suggests that the cellular ADP:ATP ratio may increase upon heat shock
(37) . It is tempting to speculate that increases in the
intracellular ADP:ATP ratio might tend to favor an ADP form of Ssa1p
and, consequently, the stable binding of substrate polypeptides.
suggesting that Ydj1p may bind to
unfolded or partially folded polypeptides. Perhaps Ydj1p enhances the
ability of Ssa1p to ``capture'' polypeptide substrates by
targeting Ssa1p to those substrates and then stabilizing the
ADP
Ssa1p
polypeptide complex.
Table: Values of K and V
for Ssalp ATPase at
several KCl concentrations
) and µM ATP
( K
).
Table: Stability of the ATPSsalp complex in
the presence and absence of effectors
slope
. These values were used to calculate rate
constants (min
) using the formula k = 0.693
t. Experiment 2 corresponds to the
data presented in Fig. 3. Experiments 1 and 2 were done on separate
days using freshly prepared or frozen
[
-
P]ATP
Ssalp complex, respectively.
Exp, experiment; ND, not determined.
-lactalbumin; BSA,
bovine serum albumin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.