(Received for publication, February 10, 1995; and in revised form, June 6, 1995)
From the
Using two independent experimental approaches to monitor
protein-protein interactions (enzyme-linked immunosorbent assay and
size exclusion high performance liquid chromatography) we describe a
general mechanism by which DnaJ modulates the binding of the DnaK
chaperone to various native protein substrates, e.g. P,
O,
, P1, RepA, as well as permanently denatured
-carboxymethylated lactalbumin. The presence of DnaJ promotes the
DnaK for efficient DnaK-substrate complex formation. ATP hydrolysis is
absolutely required for such DnaJ-dependent activation of DnaK for
binding to both native and denatured protein substrates. Although ADP
can stabilize such an activated DnaK-protein complex, it cannot
substitute for ATP in the activation reaction. In the presence of DnaJ
and ATP, DnaK possesses the affinity to different substrates which
correlates well with the affinity of DnaJ alone for these protein
substrates. Only when the affinity of the DnaJ chaperone for its
protein substrate is relatively high (e.g.
, RepA) can a tertiary complex
DnaK-substrate-DnaJ be detected. In the case that DnaJ binds weakly to
its substrate (
P,
-carboxymethylated lactalbumin), DnaJ is
only transiently associated with the DnaK-substrate complex, but the
DnaK activation reaction still occurs, albeit less efficiently.
Recently it has become clear that the Hsp70 (DnaK homologue)
activity is regulated by the DnaJ and GrpE co-chaperones (for review,
see Georgopoulos et al.(1994)). Eukaryotic Hsp40 (DnaJ
homologue) and Hsp24 (GrpE homologue) have been also identified (Silver
and Way, 1993; Bolliger et al., 1994; Lalovaya et
al., 1994). In the best studied bacterial system, where all three
heat shock proteins were first purified as host factors involved in
DNA replication (Zylicz and Georgopoulos, 1984; Zylicz et
al., 1985, 1987), DnaK's ATPase activity is stimulated up to
50-fold jointly by the DnaJ and GrpE proteins, whereas DnaJ alone
stimulates the DnaK's ATPase activity only up to 2-3-fold
(Liberek et al., 1991). Recently, the ability of the
eukaryotic Hsp40 protein to stimulate Hsp70s ATPase activity was also
shown (Cyr et al., 1992, 1994; Cyr and Douglas, 1994).
However, the role of the DnaJ homologues in functioning cooperatively
with Hsp70's is still not clear. Several groups have presented
evidence that in the presence of ATP, DnaJ inhibits the disassociation
of rhodanese, CMLA, (
)
P, and
from
DnaK (Langer et al., 1992; Hoffmann et al., 1992;
Osipiuk et al., 1993; Liberek and Georgopoulos, 1993). Another
laboratory, using the same substrate (CMLA) but a different technique,
reported that the eukaryotic DnaJ homologue (YdJ1p) and ATP are
required for the efficient release of CMLA from Hsp70 (Cyr et
al., 1992; Cyr and Douglas, 1994). Since both DnaJ and YdJ1p
interact poorly with CMLA it was proposed that if DnaJ-like proteins
had a low affinity for a polypeptide substrate, then they would be
likely to stimulate the disassociation of Hsp70 from a substrate.
However, if a DnaJ-like protein has a high affinity for a substrate it
will stabilize a Hsp70-substrate complex (Cyr et al., 1994).
Interestingly, DnaJ alone has also been shown to function as a
chaperone since it prevents other proteins from aggregation (Langer et al., 1992; Schroder et al., 1993).
Here, we
attempt to resolve some of these ambiguities in the chaperone field by
presenting a general model for the role of DnaJ in regulating
DnaK's substrate binding/release properties. Specifically, we
find that DnaJ, in an ATP-dependent reaction, inhibits the dissociation
of DnaK from its various protein substrates. This reaction occurs both
for native and denatured protein substrates, regardless of whether DnaJ
binds with high or low affinity. The ability of DnaJ to stimulate
DnaK's substrate binding properties correlates with DnaJ's
affinity for the various protein substrates. Specifically, DnaJ will
activate DnaK to bind to a substrate for which DnaJ itself has a very
low affinity (e.g. CMLA), albeit to a less extent than for
substrates to which DnaJ binds more efficiently (e.g. , RepA). During preparation of this article, a
paper was published by Szabo et al.(1994) proposing a similar
mechanism for DnaK/DnaJ synergistic chaperone action, although their
conclusion was based on binding to one denatured substrate, inactivated
luciferase. Here, by using both denatured and native protein
substrates, we show that the DnaJ-dependent activation of DnaK binding
properties is a general phenomenon.
The DnaK and DnaJ molecular chaperones, as well as all protein substrates were purified as described by Wawrzynow and Zylicz(1995). The modified ELISA and size exclusion HPLC were performed as described by Wawrzynow et al.(1995).
Figure 1:
DnaJ modulates DnaK's affinity
for its substrates. The protein substrates (indicated in the left top
corner of each panel) were preincubated (0.5 µg/well) in an ELISA
plate as described by Wawrzynow and Zylicz(1995). Following the
binding, blocking, and washing procedures, increasing amounts of DnaJ
protein in the presence of 50 ng of DnaK (in each well the
concentration of DnaK was constant) was incubated without nucleotide
(), with ATP (0.5 mM) (
), with ADP (0.5
mM) (
), with AMP-PCP (0.5 mM) (
). After 30
min at room temperature glutaraldehyde was added and the amount of DnaK
bound to its different protein substrates was estimated using anti-DnaK
serum as described by Wawrzynow and
Zylicz(1995).
ADP cannot
substitute ATP in DnaJ- and ATP-dependent binding of DnaK to the
different protein substrates (Fig. 1). In the case of
and
O only a slight stimulatory effect of ADP
was observed (Fig. 1). When AMP-PCP was used, efficient binding
of DnaK to various protein substrates was seen, regardless of whether
DnaJ was present or not (Fig. 1). Only in the case of the
P
and
substrates did the presence of ATP and high
concentrations of DnaJ stimulate the binding of DnaK to a significantly
higher level than the one observed in the absence of DnaJ, but in the
presence of AMP-PCP (Fig. 1). This result may suggest that in
these cases DnaJ increases the on rate for the DnaK-substrate complex
formation. Surprisingly, we also found that in the case of the CMLA
substrate, the absence of ATP and the presence of low concentrations of
DnaJ partially triggered the release of DnaK from CMLA (Fig. 1).
These findings were further substantiated with size exclusion HPLC (Fig. 2). Under these conditions, DnaJ does not interact with
CMLA regardless of whether ATP is present or not (Fig. 2). In
addition, when ATP was absent, we could not detect, as judged by HPLC,
a stable DnaK-DnaJ complex (Wawrzynow and Zylicz, 1995). Nevertheless,
DnaJ was able to antagonize the formation of the DnaK-CMLA complex (Fig. 2). These findings are consistent with our ELISA data
(Wawrzynow and Zylicz, 1995), suggesting the existence of a weak
interaction between DnaK and DnaJ even in the absence of ATP. In
parallel experiments, again using the HPLC method, we showed that in
the absence of ATP, contrary to the case with CMLA, DnaJ does not
release
P complexed with DnaK (result not shown). One
interpretation of these results is that, when the affinity of DnaK to
the substrate is relatively low, DnaJ could be recognized by DnaK as a
potential substrate and thus compete with the real one (CMLA).
Figure 2:
Isolation of a DnaK-CMLA complex in the
presence of DnaJ and ATP. Before injection on a Superdex 200 HPLC
column equilibrated with buffer B (Wawrzynow and Zylicz, 1995), DnaK
protein (35 µg) was incubated with CMLA (15 µg) in the absence
(CMLA, DnaK) or presence of DnaJ (40 µg) (CMLA, DnaK, DnaJ).
Control experiments, CMLA alone (CMLA), DnaJ alone (DnaJ), as well as
DnaJ preincubated with CMLA (CMLA, DnaJ), are also shown. Each
experiment was repeated in the absence (panel A) or presence
of 200 µM ATP in the premixture (panel B). The
Superdex 200 column was calibrated with the following Bio-Rad molecular
weight standards: 1, thyroglobulin (670 kDa); 2,
bovine -globulin (158 kDa); 3, chicken ovalbumin (44
kDa); 4, equine myoglobin (17.5
kDa).
When
DnaJ and ATP were simultaneously present, permanently denatured CMLA
and native P formed a complex with DnaK as judged by either ELISA
or HPLC ( Fig. 1and Fig. 2, and results not shown). The
SDS-polyacrylamide gel electrophoresis of the fraction after HPLC
indicates that DnaJ is not a part of DnaK-
P or DnaK-CMLA complexes
(results not shown). As shown in Fig. 1and Fig. 2, the
efficiency of the DnaJ-dependent activation of DnaK for binding to
unfolded CMLA or native
P is almost identical. As in the case of
P and CMLA, in the presence of DnaJ and ATP it is possible to
isolate a stable
-DnaK complex (Liberek and
Georgopoulos, 1993) and RepA-DnaK complex (Fig. 3). Very likely
due to the high affinity of DnaJ for either
or RepA
(Liberek and Georgopoulos, 1993; Wickner, 1990) in these cases it was
possible to isolate tertiary complexes, DnaK-
-DnaJ
(Liberek et al., 1993) or DnaK-RepA-DnaJ (Fig. 3;
Wickner et al., 1992; results not shown). Interestingly, in
the case of RepA even in the absence of ATP, a tertiary complex could
be detected as judged by size exclusion HPLC (Fig. 3) and
supported by SDS electrophoresis of fractions after chromatography
(results not shown). As reported by Liberek et al.(1992) in
the case of
, such a tertiary complex in the absence
of ATP was not formed. One explanation could be that
exhibits relatively high affinity toward DnaJ but low affinity
toward DnaK, whereas the RepA protein binds both chaperones equally
well (Wawrzynow and Zylicz, 1995).
Figure 3: Isolation of a tertiary DnaK-RepA-DnaJ complex. Before injection on a Superdex 200 column equilibrated with buffer B, the RepA (32 µg) was preincubated with either DnaK (35 µg) (RepA, K), or with DnaJ (40 µg) (RepA, J), or with both DnaK and DnaJ in the absence of ATP (RepA, K, J) or DnaK and DnaJ with ATP (RepA, K, J, ATP) in the premixture. In control experiments RepA was preincubated with DnaK and ATP (200 µM) in the premixture (RepA, K, ATP) and with ATP present both in the premixture and column buffer (RepA, K, ATP/ATP). The positions of RepA and DnaK alone are also shown. The Superdex 200 column was calibrated with the Bio-Rad molecular weight standards described in the the legend to Fig. 2.
To test the stability of the
DnaK-P (or DnaK-CMLA) complexes formed in the presence of DnaJ, we
preincubated these proteins with ATP for 30 min at room temperature,
then washed away the ATP and unbound proteins and added fresh buffer
with or without ATP, ADP, AMP-PCP, or ATP with DnaJ, and the reaction
was stopped with glutaraldehyde at various times. It was found that ADP
and AMP-PCP substantially stabilized the DnaK-
P or DnaK-CMLA
complexes formed under these conditions (Fig. 4). In the absence
of nucleotides the complex was not stable, while the addition of ATP
further destabilized these complexes (Fig. 4). Interestingly,
the presence of both ATP and DnaJ dramatically stabilized the
DnaK-substrate complexes compared to the situation where only ATP was
used (Fig. 4). These results suggest that the DnaJ protein may
inhibit the off rate of the DnaK-substrate complex.
Figure 4:
Stability of the DnaK-substrate complex
formed in the presence of DnaJ and ATP. P (panel A) or
CMLA (panel B) were preincubated (0.5 µg/well) in ELISA
plate wells. The wells were washed and blocked with PBS/BSA and A/BSA
as described by Wawrzynow and Zylicz(1995). Subsequently 40 ng of DnaK,
80 ng of DnaJ in buffer A/BSA supplemented with ATP (1 mM),
was added to each well and incubation proceeded for 30 min at room
temperature, followed by washing twice with 100 µl of buffer A/BSA
(time 0). After removal of ATP and unbound proteins, fresh buffer A/BSA
was added (50 µl) with: ATP (1 mM),
; ADP (1
mM),
; AMP-PCP (1 mM),
; ATP (1
mM) and DnaJ (50 ng), ▴; no nucleotide,
.
Interacting proteins were cross-linked at the indicated time by the
addition of glutaraldehyde (final concentration 0.1%). At each time
point, glutaraldehyde cross-linking proceeded for 5 min, followed by
washing (three times) with PBS/BSA. The amount of DnaK protein bound to
P or CMLA was estimated using an anti-DnaK serum as described by
Wawrzynow and Zylicz(1995).
To confirm the
requirement of ATP hydrolysis during the DnaJ-dependent formation of
DnaK-substrate complex, we preincubated DnaK and DnaJ proteins in the
presence of either ATP or ADP or in the absence of any nucleotides for
15 min at 30 °C. Following this, the reaction mixture was
immediately added onto an ELISA plate with previously immobilized
P protein and the incubation proceeded for an additional 30 min (Fig. 5A). An efficient binding of DnaK to
P was
observed only when ATP was present during preincubation of DnaK and
DnaJ molecular chaperones (Fig. 5A). Neither the
presence of ADP nor the absence of any nucleotides promotes
DnaJ-dependent binding of DnaK to
P (Fig. 5A),
suggesting that ATP hydrolysis is required for DnaJ-dependent
activation of DnaKs binding to protein substrates. Interestingly, when
DnaK, DnaJ, and ATP (or ADP) were preincubated directly on the
P-modified ELISA plate wells, similar results were obtained (Fig. 5A), suggesting that the presence of the
substrates during DnaJ- and ATP-dependent activation of DnaK is not
necessarily required. To test the effect of ADP in the stabilization of
P-DnaK complex formed during DnaJ- and ATP-dependent activation of
DnaK, we preincubated DnaK, DnaJ, and ATP (or ADP) in an Eppendorf tube
(absence of
P substrate) for 15 min at 30 °C; following this,
a 10-fold molar excess of ADP or ATP was added and the reaction mixture
was immediately placed onto an ELISA plate with previously immobilized
P protein (Fig. 5B). Surprisingly, when DnaK and
DnaJ were preincubated with ATP and added to the substrate in the
presence of excess ADP, much more efficient binding of DnaK to the
P was observed (Fig. 5B) as compared to the
situation where excess of ADP was not used (Fig. 5A).
The substitution of ATP by ADP during the first preincubation period
(activation reaction) did not lead to efficient binding of the DnaK
chaperone (Fig. 5B), thus supporting our previous
findings that ATP hydrolysis is required for DnaJ-dependent activation
of DnaK protein. The substitution of ADP by ATP during the loading of
DnaK and DnaJ onto the substrate also did not lead to an efficient
DnaK-
P complex formation (Fig. 5B). This
observation suggests that ADP is an efficient stabilizing factor of the
activated DnaK-
P complex. Similar results were obtained when DnaK,
DnaJ, and ATP or ADP were preincubated on the ELISA plate with
P
before the 10-fold molar excess of ADP (or ATP) was added (results not
shown). This again may suggest that the presence of substrate during
the DnaJ- and ATP-dependent activation of DnaK protein is not required.
Figure 5:
ATP hydrolysis is required for the
DnaJ-dependent activation of DnaK protein for binding to P.
P
was preincubated (0.5 µg/well) in ELISA plate wells. The wells were
washed and blocked with PBS/BSA and A/BSA as indicated under
``Materials and Methods.'' Panel A, an increasing
amount of DnaJ protein in the presence of a constant concentration of
DnaK protein (50 ng) was incubated for 45 min directly in the well with
ATP (250 µM,
), ADP (250 µM,
),
or without nucleotide (
). The experiment was repeated in such
way that an increasing amount of DnaJ protein with DnaK (50 ng) was
first preincubated in an Eppendorf tube for 15 min at 30 °C with
ATP (
), ADP (▴), and no nucleotide (▪). Then, the
premixture was loaded on a
P-modified ELISA plate and incubation
proceeded for additional 30 min at room temperature. The amount of DnaK
protein bound to
P was estimated as described by Wawrzynow and
Zylicz(1995), except no cross-linking with glutaraldehyde was applied. Panel B, an increasing amount of DnaJ protein in the presence
of DnaK (50 ng) was first preincubated 15 min at 30 °C in the
presence of: ATP (250 µM) (
and
) or ADP (250
µM) (
and ▴). Following this, the premixture
(50 µl) was loaded on an ELISA plate in the presence of ATP (2.5
mM) (
and
) or ADP (2.5 mM) (
and
▴) and incubation at room temperature proceeded for an additional
30 min. The amount of DnaK bound to
P was determined using
anti-DnaK serum as described by Wawrzynow and Zylicz(1995), except no
glutaraldehyde cross-linking was applied. In control experiments, an
increasing amount of DnaJ protein with a constant concentration of DnaK
was preincubated with ATP (250 µM) directly with
P in
ELISA wells for 15 min, and after addition of an excess of ADP (2.5
mM), incubation proceeded for additional 30
min.
In a control experiment, following incubation of DnaK or DnaJ with
ATP (or ADP) the proteins were separated from unbound nucleotides using
a desalting column and directly loaded onto a P-modified ELISA
plate. Only in the simultaneous presence of DnaK, DnaJ, and ATP during
the preincubation reaction was an efficient binding of DnaK protein to
P following size exclusion chromatography observed (results not
shown).
Using five different protein substrates (including denatured and native protein substrates) we were able to show that in the presence of physiological concentrations of ATP, the affinity of DnaK to its various protein substrates is correlated with the affinity of DnaJ for these protein substrates. The data presented in this paper show that regardless of the low affinity of DnaJ for CMLA (Langer et al., 1992; Wawrzynow and Zylicz, 1995), in the presence of ATP and DnaJ, a DnaK-CMLA complex readily forms. This result was verified by two independent methods, ELISA and size exclusion HPLC. Cyr and Douglas (1994) reported that in the presence of ATP and Ydj1p (an eukaryotic DnaJ homologue) CMLA was released from its complex with Ssa1p (an eukaryotic Hsp70 homologue). It is possible that the prokaryotic and eukaryotic Hsp40/Hsp70 homologues behave differently, even when the same protein substrate is used.
It was previously shown that the presence of DnaJ and GrpE accelerates up to 50-fold the hydrolysis of ATP catalyzed by DnaK (Liberek et al., 1991; Szabo et al., 1994). In addition, ATP hydrolysis is required for the efficient formation of a DnaK-DnaJ complex (Wawrzynow and Zylicz, 1995). In this paper we show that the DnaJ-dependent activation of DnaK to form a stable complex with its various protein substrates also requires ATP hydrolysis. The DnaJ-promoted activation reaction does not occur in the presence of ADP. We propose that the simultaneous presence of DnaJ and ATP hydrolysis converts the conformation of DnaK to such form which binds more efficiently to the different substrates. In agreement with this, recently published data show that the limited trypsin digestion pattern of DnaK in the presence of ATP is different, depending on the presence or absence of DnaJ (Wall et al., 1994).
The mechanism by which DnaJ, in the presence of ATP hydrolysis, induces DnaK's conformational changes remains unclear. Data presented in this paper suggest that after activation of DnaK, ATP could be depleted and the efficient DnaK's binding to the substrate is still observed. We previously found that the DnaK protein possesses an autophosphorylating activity (Zylicz et al., 1983). Sherman and Goldberg(1993) suggested that the phosphorylation of DnaK increases its affinity for a denatured substrate. Formally it is possible that DnaJ, which increases the ATPase activity of the DnaK protein, may also increase the phosphorylation of the DnaK protein. However, recently published data suggest that this is not the case: GrpE, but not DnaJ, influences DnaK's autophosphorylation (Panagiotidis et al., 1994). Another relevant question that remains to be answered is the length of time that DnaK remains in its ``active'' substrate-binding mode, following activation by DnaJ and ATP.
In this work we show
that the simple preincubation of DnaK, DnaJ, and ATP (without
substrate) is sufficient to activate DnaK to bind to P. This
result does not exclude the possibility that in situations when DnaJ
alone possesses a high affinity for the protein substrate, e.g. luciferase (Szabo et al., 1994),
(Liberek and Georgopoulos, 1993), and RepA (Wickner et
al., 1992), DnaJ besides activating DnaK may help by
``presenting'' the protein substrate to DnaK as well. In
these cases, the DnaJ-modified substrates in the presence of DnaK and
ATP would be converted into a stable tertiary DnaK-substrate-DnaJ
complex. In such complexes, the DnaK and DnaJ chaperones may both bind
the common protein substrates.
In all other cases we examined, where
DnaJ possesses a relatively low affinity toward the substrate (O,
P, and CMLA), the presence of DnaJ accelerates the ATP hydrolysis
and the formation of a stable substrate-DnaK complex, with which DnaJ
interacts only transiently. Recently Liberek et al.(1995)
reported that a truncated DnaJ protein (DnaJ12), which lacks the DnaJ
peptide binding site but still accelerates DnaK's ATPase
activity, can still promote the ATP-dependent formation of a stable
DnaK-
complex, but itself is not a part of this
complex. The possibility that in some cases DnaJ can work catalytically
to promote the formation of a DnaK-substrate complex not only does not
contradict, but actually is a substantial extension of the model
recently proposed by Szabo et al.(1994). In biological terms
what our findings imply is that when DnaJ binds tightly to a protein
substrate, this may lead to the formation of a DnaK-substrate-DnaJ
complex. When DnaJ does not bind efficiently to a substrate, it can
still catalyze the formation of the DnaK-substrate complex.
The DnaK-substrate-DnaJ complex is substantially stabilized by ADP. These complexes are destabilized in the absence of nucleotides, and to an even greater extent in the presence of ATP. These results suggest that the induction of conformational changes in DnaK during the release of nucleotides or the exchange of nucleotides to a different form (i.e. ADP to ATP) triggers the release of DnaK from its substrate. Only the presence of ADP, where putative exchange of nucleotides will not induce conformational changes of DnaK, does not destabilize the DnaK-substrate complex. A similar effect of ADP in the stabilization of Hsp70 bound to different substrates has been previously described by other investigators (Prasad et al., 1994; Palleros et al., 1994).