(Received for publication, March 25, 1994; and in revised form, November 11, 1994)
From the
In previous work we found that bovine brain hsp70 has a single binding site for nucleotide, and that, with ATP at this site, the rates of association and dissociation of clathrin from hsp70 are fast, whereas with ADP at this site, these rates are unmeasurably slow. In the present study we show, first, that peptide C, cytochrome c peptide, and RNase S peptide bind competitively with clathrin, suggesting that they bind to the same site on hsp70, although RNase S peptide binds an order of magnitude more weakly than peptide C and cytochrome c peptide. Second, we show that, with ADP bound to hsp70, as occurs with clathrin, the rate constant for dissociation of peptide markedly decreases compared to the rate constant observed in ATP. In contrast, ADP only slightly decreases the rate of association of peptide. Based on these data we propose a model in which substrates of hsp70 bind to and dissociate from the ATP form of the enzyme, while, following ATP hydrolysis, they are locked onto the ADP form of the enzyme, unable to dissociate until ADP is released and ATP rebinds.
The 70-kDa class of heat shock proteins (hsp70) ()is
one of the most prominent sets of proteins produced during heat shock
and has been observed in a wide variety of species from Escherichia
coli to man (Lindquist and Craig, 1988; Nover and Scharf, 1991).
Many members of the hsp70 family are produced constitutively by the
cell and are essential for cell viability. These proteins act as
molecular chaperones, with involvement in such diverse functions as the
folding and unfolding of proteins, the monomerization of protein
complexes, and the translocation of proteins into lysosomes, the
endoplasmic reticulum, mitochondria, and the nucleus (Ellis and Van der
Vies, 1991; Gething and Sambrook, 1992; Hendrick and Hartl, 1993).
Given their general role as molecular chaperones, it is not surprising that the hsp70s have been shown to interact with a wide variety of proteins both in vivo and in vitro. For example, Beckmann et al.(1990) suggested that constitutive cytoplasmic hsp70 binds to almost all proteins as they are being synthesized in vivo, while, in vitro, Palleros et al.(1991) showed that cytoplasmic hsp70 forms stable complexes with a variety of unfolded proteins but not with their properly folded counterparts. In addition, there is the well characterized interaction of bovine brain hsp70 with clathrin (Schmid and Rothman, 1985; Prasad et al., 1994) and clathrin coated vesicles (Schlossman et al., 1984; Greene and Eisenberg, 1990).
Given the ubiquitous
interactions of the hsp70 proteins, it might be expected that these
proteins would also interact with peptides. Flynn et al.(1989)
have shown that peptide C ()and peptide A, both of which are
derived from vesicular stomatitis virus glycoprotein, interact with
cytoplasmic hsp70 (Flynn et al., 1991). RNase S peptide and a
smaller peptide derived from it, KFERQ, were discovered to interact
with hsp70 by Dice and co-workers in their study of lysosomal
degradation of specific cytosolic proteins (Chiang et al.,
1989, Terlecky et al., 1992). Another peptide, a peptide
fragment of pigeon cytochrome c (Vanbuskirk et al.,
1989), was discovered to interact with hsp70 in a study which suggested
that a member of the hsp70 family is involved in the transport of
processed antigen from acidic vesicles to the surface of antigen
presenting cells.
It is important to understand the mechanism of interaction of hsp70 with its substrates. We previously found that with ATP bound to hsp70, there was rapid association and dissociation of clathrin, but with bound ADP both of these rates decreased to almost zero (Prasad et al., 1994). Despite this profound effect on rates, the bound nucleotide had only a small effect on the affinity of clathrin for hsp70. On the other hand, the binding of peptides appears to be much stronger in the presence of ADP than ATP. Flynn et al. (1989) found that the dissociation constants for ATPase activation by peptides A and C (determined in ATP) were much higher than the dissociation constants determined in ADP. Similarly, Landry et al.(1992) reported that the affinity of peptide C for DnaK is greatly increased in the presence of ADP.
In the present study we investigated the interaction of several peptides with bovine brain hsp70. We found that the peptide substrates and clathrin appear to bind to the same site on hsp70. The effects of replacing ATP with ADP on both rates of association and binding strength were somewhat variable. However, in all cases replacing ATP with ADP markedly decreased the rate of dissociation of substrate from hsp70. Furthermore, we found that it is ATP binding rather than ATP hydrolysis which causes dissociation of peptide. On the basis of these results we propose a model in which substrates are locked on hsp70 with bound ADP, but not on hsp70 with bound ATP.
Clathrin-coated vesicles, clathrin, and bovine brain hsp70
were prepared and their concentrations were determined as described in
Prasad et al.(1993). The yeast hsp70 protein, ssa1p, was
purified from strain MW141 (Werner-Washburne et al., 1987)
according to the procedure of Gao et al.(1991). Prepared brain
hsp70 has 1 mol of ADP/mol of hsp70 (Gao et al., 1993). hsp70
with bound [C]ADP was prepared by exchange of
nucleotide (Gao et al., 1993). Hsp70 and peptide were
trace-labeled by reductive alkylation using NaCNBH
and
[
C]formaldehyde as described by Jentoft and
Dearborn (1983).
The solution used in the experiments and during
column chromatography was buffer A (20 mM imidazole, 25 mM KCl, 10 mM ammonium sulfate, 2 mM magnesium
acetate, and 1 mM dithiothreitol, at pH 7.0). The nucleotide
conditions in the experiments were pure ATP (1 mM ATP, 30
units/ml creatine phosphokinase, 15 mM creatine phosphate), or
pure ADP (100 µM ADP, 50 units/ml hexokinase, and 5 mM glucose), or 90% ADP, 10% ATP (0.9 mM ADP, 0.1 mM ATP, and 1 mM P).
Uncoating of coated
vesicles was measured according to the procedure described by Greene
and Eisenberg(1990). ATPase activity of hsp70 at 25 °C was measured
with [-
P]ATP as described previously (Chock
and Eisenberg(1979). Studies involving the binding or rate of binding
of peptide to hsp70 were performed using chromatography to separate the
free and bound peptide. In the case of competitive binding studies of
peptide with clathrin the reaction mixture was applied to a Superose 6
column (HR10/30) using a Pharmacia FPLC system as described in Prasad et al.(1993). In measuring the binding and rate of binding
peptide to hsp70, the reaction mixture was generally applied to a
Superose 12 column (HR10/30) using a Pharmacia Biotech Inc. FPLC system
to separate the free and bound peptide. The Superose 6 and Superose 12
columns were equilibrated and eluted in buffer A containing 1 mM ADP and 1 mM P
. During chromatography, less
than 5% of the peptide is dissociated from the hsp70, based on the rate
of peptide release from hsp70 in ADP at 4 °C being an order of
magnitude slower than the rate at 25 °C. (
)In the
experiment measuring the rate of dissociation of peptide and nucleotide
from hsp70 in ATP, the free nucleotide or free peptide was separated
from the hsp70-nucleotide-peptide complex by spinning samples through
Sephadex G-50 spin columns as described by Penefsky(1977). Controls
showed that more than 80% of the protein was recovered, while more than
98% of the free nucleotide and free peptide was retained on the column.
MES, creatine phosphokinase, creatine phosphate, ATP-agarose, RNase
S peptide, and hexokinase were from Sigma.
[C]Formaldehyde and
[
-
P]ATP were from NEN-DuPont and
[
C]ADP was from ICN. The G-50 spin columns were
from 5 Prime
3 Prime, Inc. The concentration of peptides was
based on the protein content determined from amino acid analysis.
Figure 1:
Inhibition of
clathrin uncoating by peptides. Hsp70 (0.6 µM) was
preincubated with peptide and 0.5 µM coated vesicles for
30 min at 25 °C. At time 0, 1 mM ATP was added and the
amount of clathrin released was then determined. These experiments were
performed in the absence of peptide () or in the presence of
peptide as follows: 1 mM cytochrome c peptide
(
), 0.2 mM cytochrome c peptide (
), 1
mM peptide C (
), 0.3 mM peptide C (
), 1
mM KFERQ (
), 1 mM RNase S peptide
(
).
The inhibition of
uncoating by peptide C and cytochrome c peptide suggested that
they might bind at the same site as clathrin on hsp70. To verify this,
we performed competitive binding studies between purified clathrin and
peptide in ATP. Fig. 2, A and B, shows the
results of these experiments for cytochrome c peptide and
peptide C, respectively. In both cases, the plots are consistent with
competitive binding, and the abscissa intercepts of the derivative
plots yielded K of about 300 µM. In
contrast to peptide C and cytochrome c peptide, neither RNase
S peptide nor KFERQ caused any significant displacement of the clathrin
from hsp70 at 1 mM peptide concentration (data not shown).
Figure 2:
The competitive binding of peptide and
clathrin to hsp70 in ATP obtained with either cytochrome c peptide (A) or peptide C (B). Varying
concentrations of clathrin (3-10 µM) were mixed with
1 µMC-labeled hsp70 for 30 min at 25 °C.
This was done in the absence of peptide (
), or in the presence of
0.25 (
), 0.5 (
), or 1 mM (
) peptide. The
data were plotted on a double reciprocal plot of fraction bound versus clathrin concentration. The inset in each
panel is a derivative plot of slope versus peptide
concentration to obtain the K
from the
abscissa intercept.
If the K values obtained for the peptides by
competition with clathrin are valid, similar dissociation constants
should be obtained in direct binding studies of the peptides.
Unfortunately, even using radioactively labeled peptides, it was not
possible to measure these binding constants accurately by direct
binding studies because the binding is too weak in ATP. However,
Rothman and co-workers (Flynn et al., 1989) previously showed
that several peptides including peptide C activated the ATPase activity
of hsp70. If peptide C and cytochrome c peptide significantly
activate the ATPase activity of hsp70, then the K
values determined from half-maximal stimulation of the ATPase
activity should be identical to the K
values
obtained from the above competition studies.
To obtain linear plots
in these experiments, the enzyme was preincubated with ATP for 7 min to
replace ADP initially present at the active site of the enzyme (Gao et al., 1993a) and the experiments were carried out at rather
high ATP concentration to avoid product inhibition which began to occur
when a small fraction of the ATP was hydrolyzed. Both peptide C and
cytochrome c peptide caused significant activation of the
hsp70 ATPase activity while 1 mM RNase S and KFERQ caused
considerably less activation (Fig. 3A). With peptide C
and cytochrome c peptide, we were able to obtain values for V and K
by measuring the
hsp70 ATPase activity over a wide range of peptide concentrations (Fig. 3B). The V
values were 2
10
s
and 1.3
10
s
for cytochrome c peptide and peptide C, respectively, showing a maximum of 5-fold
activation of the hsp70 ATPase activity. The K
values were 200 and 250 µM for cytochrome c peptide and peptide C, respectively, in good agreement with the
values obtained from the competition studies (Fig. 2).
Figure 3:
ATPase activation of hsp70 by peptides.
Hsp70 (10 µM) was preincubated with 200 µM [-
P]ATP for 7 min at 25 °C. At
zero time, peptide was added. In A, a plot of P
release versus time, the data were obtained in the
absence of peptide (
), in the presence of 1 mM peptide C
(
); 0.5 mM peptide C (
); 1 mM cytochrome c peptide (
), 0.3 mM cytochrome c peptide (
), 1 mM RNase S peptide (
);
and 1 mM KFERQ (
). In B, the ATPase data were
plotted on a double reciprocal plot of hsp70 ATPase activity versus peptide concentration obtained by adding varying concentrations
(0.2-2.0 mM) of either cytochrome c peptide
(
) or peptide C (
) to hsp70. The data were corrected for the
rate of ATP hydrolysis by the hsp70 in the absence of
peptide.
Figure 4:
Scatchard plot of the binding of
cytochrome c peptide to brain hsp70 and ssa1p in 90% ADP, 10%
ATP. With brain hsp70 (), varying concentrations of
C-labeled peptide (3-15 µM) were
incubated with 5 µM hsp70 for 2 h at 25 °C. With ssa1p
(
), varying concentrations of
C-labeled peptide
(1-10 µM) were incubated with 3 µM ssa1p for 2 h at 25 °C.
To determine if the effect of nucleotide on the binding strength of
peptide depends on the nature of the hsp70, we measured the binding
strength of labeled cytochrome c peptide to ssa1p, a
cytoplasmic yeast hsp70, in both ATP and in 90% ADP, 10% ATP. In both
cases we obtained linear Scatchard plots which yielded a K of 2 µM in 90% ADP, 10% ATP (Fig. 4, solid triangles), and a K
of 25 µM in ATP (data not shown), in both cases
stronger binding than occurs to bovine brain hsp70. These data suggest
that the effect of nucleotide on the binding strength of substrates to
hsp70 is rather variable. With bovine brain hsp70, clathrin binds
4-fold stronger and peptide binds 40-fold stronger in ADP than in ATP
while with yeast ssa1p peptide binds 12-fold stronger in ADP than in
ATP. It should also be noted that ssa1p differs from hsp70 in that
peptide does not significantly activate the ATPase activity of
ssa1p.
Since ADP strengthens the binding of peptide to bovine brain hsp70 40-fold, it seemed possible that in 90% ADP, 10% ATP we could detect the binding of KFERQ and RNase S peptide. We find that RNase S peptide competes with the binding of both clathrin and cytochrome c peptide (data not shown), although in similar experiments, no evidence could be obtain for the binding of KFERQ. The dissociation constant obtained from these competition experiments is about 200 µM, more than an order of magnitude weaker than the binding of cytochrome c peptide, peptide C, and clathrin under the same conditions. Direct binding studies in which RNase S peptide was labeled by reductive methylation confirmed that the dissociation constant was about 150 µM (data not shown). These data suggest that RNase S peptide may indeed bind to the same site on hsp70 as clathrin, peptide C, and cytochrome c peptide but much more weakly than the binding of these other three substrates.
Figure 5:
Dissociation rate of cytochrome c peptide from brain hsp70 and ssa1p. In A, the
dissociation rate of peptide from hsp70 (,
) and ssa1p
(
) was measured by exchange in either pure ADP (
,
)
or 90% ADP, 10% ATP (
). This was done by either mixing 10
µM brain hsp70 and 60 µM cytochrome c peptide for 2 h at 25 °C, followed by the addition of 5
µM
C-labeled cytochrome c peptide or
by mixing 3 µM ssa1p with 25 µM peptide in
ADP for 2 h at 25 °C, followed by the addition of 3 µM
C-labeled peptide. In B, the dissociation
rate of cytochrome c peptide in ATP was measured for ssa1p by
exchange, and for brain hsp70, by determining the difference in the
rate of ADP release (
) and the rate of peptide release (
)
from the hsp70-ADP-peptide complex. ADP release was measured by
incubating 100 µM cytochrome c peptide with 5
µM [
C]ADP-hsp70 for 2 h at 25
°C followed by addition of 1 mM ATP. Peptide release was
measured by incubating 10 µM
C-labeled
peptide with 10 µM hsp70 for 2 h at 25 °C followed by
addition of 1 mM ATP. In both cases samples were then
centrifuged through spin columns at the indicated times. The rate of
release of peptide from ssa1p in ATP (
) was measured by
incubating 15 µM ssa1p with 100 µM peptide in
1 mM ATP for 30 min at 25 °C, followed by the addition of
10 µM
C-labeled
peptide.
We next determined if, as with clathrin, the rate of dissociation of
peptide increases in ATP compared to ADP. Since peptide binds very
weakly to enzyme in the presence of ATP under equilibrium conditions,
it is not possible to obtain enough enzyme-ATP-peptide complex relative
to free peptide to directly measure by exchange experiments the rate of
dissociation of peptide from the enzyme-ATP complex. Therefore, our
approach was to prepare the enzyme-ADP-peptide complex and then
determine the rate of dissociation of peptide from enzyme-ATP using a
two-step approach. First, we determined the rate at which ADP was
released from the enzyme-ADP-peptide complex after addition of excess
ATP. We then determined the overall rate that peptide was released from
the enzyme-ADP-peptide complex after addition of excess ATP. Since we
have already shown that peptide dissociates very slowly from the
enzyme-ADP-peptide complex, the difference between these two rates
should provide a measure of the rate of peptide release from the
enzyme-ATP-peptide complex. Since the rates of both ADP release and
peptide release from the enzyme-ADP-peptide complex were very rapid,
these experiments were carried out using spin columns rather than FPLC
chromatography (see ``Materials and Methods''). We found
that, following addition of excess ATP, ADP dissociated from the
hsp70-peptide-[C]ADP complex with a half-life of
about 65 s (Fig. 5B, open circles) while
C-labeled peptide dissociated with a half-life of about 80
s (Fig. 5B, closed circles). Therefore peptide
appears to dissociate from the hsp70-ATP complex with a half-life of
about 15 s. This yields a rate constant for dissociation of peptide in
ATP of 4.6
10
s
, more
than 200-fold faster than the rate constant determined in ADP. This is
qualitatively similar to the marked increase in the rate of
dissociation of clathrin in ATP compared to ADP.
Since ADP and ATP affected the binding strength of cytochrome c peptide to yeast ssa1p much less than its binding strength to bovine brain hsp70, we were interested in determining the effect of ATP and ADP on the rate of dissociation of the cytochrome c peptide from yeast ssa1p. Cytochrome c peptide binds much more strongly to yeast ssa1p than to bovine brain hsp70 in ATP. Therefore, using the exchange method we employed for the cytochrome c peptide-hsp70 complex in ADP (Fig. 5A), we could directly determine the rate of peptide release from the ssa1p-cytochrome c peptide complex in both ADP and ATP. Our results show that the half-life for dissociation of peptide from the peptide-ssa1p-ADP complex is 67 min (Fig. 5A, solid triangles), while the half-life for dissociation of peptide-ssa1p-ATP complex is 2.5 min (Fig. 5B, solid triangles).
Since the
presence of ADP at the active site of the enzyme markedly inhibits the
dissociation of peptide from the enzyme, it seems very unlikely that
hydrolysis of ATP to ADP and P would be required for
peptide dissociation. To test this point we prepared
C-labeled peptide-hsp70-ADP complex, removed all free
nucleotide, and then added a 10-fold excess of
[
-
P]ATP. Fig. 6shows that, as
expected, there was no burst of ATP hydrolysis associated with peptide
dissociation. Over a 2-min period 2.8 µM enzyme-peptide
complex dissociated while only about 0.25 µM ATP was
hydrolyzed. Therefore, it is ATP binding rather than ATP hydrolysis
which dissociates the enzyme-peptide complex.
Figure 6:
Comparison of the rate of release of bound
peptide and the rate of ATP hydrolysis of the hsp70 peptide complex.
Hsp70 with bound stoichiometric C-labeled cytochrome c peptide was prepared by mixing 50 µM hsp70 with 50
µM
C-labeled peptide in 90% ADP, 10% ATP.
After 2 h at 25 °C, the solution was run on a Pharmacia PD-10
column to remove free nucleotide. hsp70-peptide complex (4
µM) was then mixed with either 40 µM ATP or
[
-
P]ATP to measure bound peptide (
) or
ATPase activity (
), respectively.
Figure 7:
Time course of binding of cytochrome c peptide to hsp70 in the presence of nucleotide. The rate of
binding was measured using 5 µM hsp70 and 50 µMC-labeled cytochrome c peptide in the
presence of different nucleotides at 25 °C. In Fig. 7A, the rate of binding was measured in the
presence of ATP, while in Fig. 7B the rate of binding
was measured in the presence of pure ADP (
) or in the presence of
90% ADP, 10% ATP (
).
The rate
constant for association in ATP can also be calculated from the
dissociation constant for cytochrome c peptide-hsp70 complex
in ATP determined in Fig. 3(200 µM) and the rate
constant for dissociation in ATP determined in Fig. 5B (4.6 10
s
) which
yields a value for the rate constant of association of 230 M
s
, in good agreement
with the 140 M
s
determined by directly measuring the rate of association. It
should be noted that the rate constant for dissociation which we
measured in Fig. 5B was for dissociation of peptide
from the pure ATP bound state while the dissociation constant was
measured under steady-state conditions where only a fraction of the
enzyme may be in the ATP bound state. Correction for this point would
decrease the rate constant for dissociation under steady-state
conditions and therefore would also decrease the calculated value for
the rate constant for association. For example if half of the enzyme is
in the ATP-bound state during steady-state ATP hydrolysis as we have
recently found,
the above calculation would yield a value
for the rate constant of association of approximately 115 M
s
rather than 230 M
s
.
We were next
interested in determining whether the rate of association of peptide
with hsp70 decreases in ADP compared to ATP. We found that peptide
binds to the enzyme with a half-life of about 12 min at a concentration
of 50 µM peptide (Fig. 7B). This yields a
rate constant of 20 M s
for association of peptide with enzyme which is about one-seventh
the rate constant for association measured in ATP. As with the rate
constant for dissociation, nearly the same rate constant for
association was obtained in pure ADP and 90% ADP, 10% ATP. Note,
however, that the rate constant for association was slightly slower in
ADP than in 90% ADP, 10% ATP, probably because of the slow equilibrium
between monomeric and polymeric hsp70 which occurs in ADP (Schmid et al., 1985). Having determined both the rate constants for
association and dissociation in 90% ADP, 10% ATP, we could calculate
the binding constant from the ratio of rate constants. This yields a
value of 8 µM which is in good agreement with the binding
constants determined by competition of peptide with clathrin and by
direct binding experiments.
If one can determine the major effect that ATP and ADP have on the properties of an ATPase, it often leads to an important clue about the basic mechanism of action of the enzyme. For example, myosin, the ATPase which drives muscle contraction, binds 5 orders of magnitude more strongly to actin with ADP than with ATP at the active site; it is the transition from the weak-binding to the strong-binding state which apparently drives muscle contraction (Eisenberg and Hill, 1985; Rayment et al., 1993). Another example is the various Gproteins which also occur in two major conformational states determined by their bound nucleotide. Our previous determination that hsp70 has only one site for nucleotide (Gao et al., 1993) along with our investigation of the interaction of hsp70 with clathrin (Prasad et al., 1994), suggested that hsp70 may also occur in two major conformational states which differ, not in the binding strength of clathrin, but in the rates of formation and dissociation of the hsp70-clathrin complex; in ATP association and dissociation of the clathrin are very fast while in ADP the rates of both association and dissociation become too slow to measure. In the present study we investigated the interaction of several peptide substrates with hsp70 to determine first, if they bind to the same site as clathrin, and second, if they show a similar response to ATP and ADP.
Although various proteins and peptides apparently bind to the same site on hsp70, they do so with widely different affinities. We found that RNase S and KFERQ bind to hsp70 much more weakly than the other peptides used in this study. The dissociation constant we obtained for RNase S peptide in ADP was an order of magnitude larger than that reported by Terlecky et al.(1992), Huang et al.(1993), and Wang et al.(1993), which were measured in the absence of added nucleotide. However, in these studies, either substrate or enzyme were linked to a solid support. Another substrate, deoxysporogualin, has also been reported to bind to hsp70 based on its binding to a deoxysporogualin-linked Sepharose column (Nadler et al., 1992). However, using competition with peptide binding as an assay for binding, we have not been able to substantiate the binding of this substrate to hsp70, even in ADP (data not shown). It may be that when binding is measured by using a technique where substrate or enzyme is linked to a solid support, stronger binding is observed than occurs in solution. This might particularly be the case with hsp70 because it has been found to bind directly to glutathione S-transferase-Sepharose columns (Wang et al., 1993).
In contrast to the effect of nucleotide on the off-rate of substrate, its effects on both the on-rate and the strength of binding were more variable. With regard to the on-rate, ADP, compared to ATP, decreased the rate of clathrin binding to bovine brain hsp70 by orders of magnitude, but decreased the rate of peptide binding to bovine brain hsp70 by a factor of 7 and decreased the rate of peptide binding to yeast ssa1p by only a factor of 2. Likewise the binding of clathrin to bovine brain hsp70 increased 4-fold, the binding of cytochrome c peptide to ssa1p increased 12-fold, and the binding of peptides to bovine brain hsp70 increased 40-fold in ADP compared to ATP. From these data we conclude that the largest consistent difference caused by nucleotide in the interaction of substrates with hsp70 is a marked decrease in the rate of dissociation in ADP with the half-life for dissociation being equal to or less than 2.5 min in ATP and more than 60 min in ADP in all cases.
We observed one other interesting difference in the effect of ADP on the rates of dissociation of clathrin and peptides from hsp70. In 90% ADP, 10% ATP, the rate of clathrin dissociation is considerably faster than in pure ADP where it is too slow to be measured. On the other hand, there is almost no difference in the rate of dissociation of peptide from the enzyme-peptide complex in ADP and 90% ADP, 10% ATP. This is probably because with clathrin, the rate of dissociation is so slow from the hsp70-ADP complex that when ATP is added, the path of dissociation involving exchange of ADP with ATP is faster than the rate of direct dissociation. On the other hand with peptide, the rate of direct dissociation although slow, is much faster than with clathrin, and therefore dissociation involving exchange of ADP with ATP is not significantly faster than the rate of direct dissociation.
Figure 8: Model of hsp70 action. The cycle shows that the hsp70 (E) picks up and delivers protein (P), presumably at different sites, when ATP is bound to the enzyme. When ADP is bound, the protein is trapped on the enzyme for transport.
This simple model of hsp70 action fits nicely with the physiological role of the hsp70 proteins in which, in their role as chaperones, they carry proteins from one point in the cell to another. For example, in their involvement in the transport of proteins across the mitochondrial membrane, it has been suggested that cytoplasmic hsp70 binds to an unfolded protein at the ribosome and then releases this unfolded protein at the outside of the mitochondrial membrane. After passing through the membrane, mitochondrial hsp70 then binds unfolded protein at the inner membrane of mitochondria and transfers it to the 60-kDa chaperonin protein which folds it (Craig, 1993). Cofactors which control the rate of ATP hydrolysis may be involved in the initial attachment of proteins to the hsp70 while other cofactors may prevent premature release of ADP before the hsp70 reaches its proper destination and, conversely, induce ADP release at the point where the protein substrates are delivered.
Recently a somewhat different model of hsp70 action was proposed suggesting that substrates bind to the ADP form of the enzyme rather than to the ATP form of the enzyme which was considered to be inactive; in this model ATP hydrolysis occurred after substrate dissociated from the ATP form of the enzyme (Palleros et al., 1993). A major difficulty with this model is that it cannot explain how peptides and other substrates of hps70 activate the ATPase activity of the enzyme. Since, in the absence of substrate, at least 85% of the enzyme occurs in the ATP form (Gao et al., 1993), the ATP hydrolysis step must be the rate-limiting step in the ATPase cycle. Therefore, if this step occurs after peptide or protein substrates dissociate from the enzyme, it is difficult to see how these substrates could activate the ATPase activity of the enzyme as we (Fig. 3) and others (Flynn et al., 1989; Sadis and Hightower, 1992; Palleros et al., 1993; Blond-Elguindi et al., 1993) have observed experimentally. In contrast, in our model substrate binds to the ATP form of the enzyme and therefore can directly increase the rate of the ATP hydrolysis step and in this way activate the ATPase activity of the enzyme.
In studying the relatively nonspecific interaction of peptides with hsp70 in vitro, it must be kept in mind that this interaction may mimic only part of the in vivo reaction cycle of the enzyme with its natural substrates and participating cofactors. We recently obtained evidence that a cofactor is required for the uncoating reaction to occur (Prasad et al., 1993), and under certain conditions the presence of both clathrin baskets and cofactor can cause 100-fold activation of the ATPase activity of hsp70 (Barouch et al., 1993), much more than occurs with the peptide substrates investigated in this study. The fact that clathrin-coated vesicles are the true in vivo substrate of hsp70 may explain why much higher ATPase activities are observed with clathrin baskets and cofactor than with peptide substrates and also why the effects of ATP and ADP we observed on the rates of clathrin association and dissociation are more dramatic than we find with the less specific peptide substrates.