From the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, April 6, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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In addition to promoting protein folding and
translocation, molecular chaperones of Hsp70/DnaJ families are
essential for the selective breakdown of many unfolded proteins. It has
been proposed that chaperones function in degradation to maintain the substrates in a soluble form. In Escherichia coli, a
nonsecreted alkaline phosphatase mutant that lacks its signal sequence
(PhoA All cells utilize energy-dependent proteolytic
pathways to degrade selectively polypeptides with highly abnormal
conformations (1, 2). Such misfolded proteins may result from nonsense or missense mutations, mistakes in translation, genetic engineering (e.g. gene fusions), failure of a polypeptide subunit to
associate with others, or intracellular denaturation (3, 4). Another way such abnormal proteins may appear in cells is through incorrect localization; for example, if a secretory protein is retained in the
cytosol, where it cannot form the correct disulfide bonds due to the
reducing environment (5), the protein is rapidly degraded (6). It
remains unclear how the cell's proteolytic apparatus recognizes and
destroys such abnormal polypeptides while sparing normal cell constituents.
Molecular chaperones have been implicated in this degradative process
in both bacterial and eukaryotic cells (7, 8). In addition to
participating in the folding of nascent polypeptides, their
translocation across membranes, and the assembly of oligomeric complexes (9, 10), molecular chaperones of the Hsp70/DnaK family and
the cofactors of the DnaJ families have recently been shown to play an
essential role in protein degradation (6, 11, 12). A characteristic
feature of Hsp70/DnaK is its ability to bind selectively to hydrophobic
oligopeptides or unfolded proteins in extended conformations, where
such domains may be exposed (13-15). DnaJ stimulates the ATPase
activity of DnaK (16, 17) and by itself can also bind to certain
unfolded proteins (18-21). Both components can thus possibly
facilitate the recognition of substrate conformations or act as
cofactors in the degradative process.
Among the first examples of the involvement of DnaJ/DnaK in the
degradation of a specific abnormal protein was alkaline phosphatase (PhoA61), which is not secreted from the cytosol due to a missense mutation in its signal sequence (6). Its rapid degradation at 37 °C
is mediated in part by the ATP-dependent protease La (Lon)
and also requires DnaK, because a dnaK-null mutant
completely prevents its breakdown. Interestingly, dnaK756, a
missense mutation that fails to release substrates in response to ATP,
was found to enhance PhoA61 degradation, whereas a dnaJ
mutant, which reduced PhoA61 association with DnaK, slowed its
turnover. Thus, complex formation between PhoA61 and DnaK appears
essential for its rapid degradation. Presumably, the successful folding
or secretion of a normal polypeptide involves only transient
association with DnaK. We therefore proposed that the chaperone,
through its prolonged association with a nonfoldable protein, may
trigger proteolysis, perhaps by helping to stabilize an abnormal
polypeptide in an unfolded, extended conformation that favors
recognition or digestion by the protease.
One characteristic feature of unfolded proteins is their tendency to
aggregate, and an important function of molecular chaperones of the
Hsp70 and DnaJ families is to prevent this process in vivo (15, 22). In addition, DnaK, DnaJ, and GrpE, together with ClpB, can
promote the solubilization of aggregated proteins (23, 24) in
Escherichia coli, as can their homologs in yeast. It has
been suggested that the primary function of DnaJ/DnaK in proteolysis is
to help maintain unfolded proteins in a soluble form, which should
render them more susceptible to proteolytic attack (25). In several
cases, undegraded abnormal proteins have been found to accumulate as
intracellular aggregates. For example, in E. coli, the
degradation of the short-lived transcription factor, RcsA, by protease
La was retarded in a dnaJ mutant, where a significant fraction of RcsA was found in particulate form (26). Similarly, in
yeast mitochondria, the degradation by the ATP-dependent
protease, Pim1 (a homolog of La), of two fusion proteins, bovine
lactalbumin and cytochrome b2-DHFR, requires the
DnaK homolog mt-Hsp70 and the DnaJ homolog Mdj1 (11). In the absence of
Mdj1, these fusion proteins formed insoluble aggregates. However, in
these chaperone-deficient cells, there was no evidence that the
aggregation of these substrates actually caused the block in
proteolysis and was not a consequence of the reduced degradation. In
other words, if the chaperones are required directly in the degradative
process, when degradation is blocked in the chaperone mutants, the
unfolded polypeptides that accumulate may then tend to aggregate. Thus,
establishing direct involvement of DnaK/DnaJ (Hsp70/40) in the
proteolytic pathway is difficult, because the association of these
chaperones with the substrate may be important both for facilitating
proteolytic attack and for promoting the proper folding of the substrate.
To distinguish these possibilities and to clarify the role of the
chaperones in the breakdown of unfolded proteins, we have studied the
cytosolic degradation of a nonsecreted alkaline phosphatase lacking the
entire signal sequence (PhoA Materials and Strains--
The E. coli strains used
in this study are listed in Table I. All strains are isogenic,
constructed by P1vir transduction into the WP551 host strain
(a kind gift of Dr. W. Prinz) according to standard bacterial genetics
procedures (30). Cells were grown and maintained in M9 minimal medium
supplemented with essential amino acids, thiamine, 0.5% glucose, and
appropriate antibiotics at 30 °C. The polyclonal antibody against
PhoA was obtained from 5 Prime Assays of PhoA Degradation--
The rate of PhoA degradation was
measured either by Western blot analysis or by a pulse-chase and
immunoprecipitation protocol as described before (6, 31). Both
approaches gave similar results, and therefore only the Western blot
method was routinely used. Cells were grown at 30 °C to
mid-logarithmic phase and then shifted to 37 °C in the presence of 2 mM isopropyl-1-thio- Assays of PhoA Activity--
Assay of alkaline phosphatase
activity was performed using a procedure slightly modified from the one
described previously (27, 29). After the induction of PhoA expression
at 37 °C as described above, cell cultures in duplicates were
incubated on ice for 20 min in the presence of 100 mM
iodoacetamide, which prevents possible spontaneous renaturation of
PhoA( Immunoprecipitation--
After the induction of PhoA expression
at 37 °C (as described above), cells were harvested, resuspended in
50 mM Tris-HCl (pH 7.5), 5 mM EDTA, and 100 mM KCl, and disrupted by sonication. A soluble extract was
obtained by taking the supernatant after centrifugation at 15,000 × g for 15 min. Immunoprecipitation of PhoA was carried out
by the addition of an anti-PhoA antibody to the soluble extract for
1 h on ice, followed by the addition of bovine serum
albumin-coated protein A-Trisacryl beads. The mixture was rotated at
4 °C for an additional 30 min and washed extensively with the same
buffer. Precipitates were resolved on 12% SDS-PAGE and transferred
onto a nitrocellulose membrane for Western blot analysis. The amount of
PhoA precipitated was confirmed by anti-PhoA antibody, and the presence
of DnaJ and proteases La and ClpA that coprecipitated with PhoA was
detected with their respective antibodies, followed by
125I-protein A. Quantitation of band intensity was
performed on a PhosphorImager.
Fractionation of Cell Lysates--
As described for
immunoprecipitation, cells were disrupted by sonication to yield total
cell lysates. Centrifugation of the total lysate at 15,000 × g for 15 min produced the low speed pellet and supernatant
fractions. The supernatant was subsequently subjected to
ultracentrifugation at 100,000 × g for 1 h to
give the high speed pellet and supernatant fractions. The pellets from
both centrifugation steps were resuspended in the same buffer and in the same volume as the recovered supernatants. Equal volumes of the
fractions were subjected to SDS-PAGE, and the amount of PhoA present in
each fraction was detected by Western blot with an anti-PhoA antibody
and 125I-protein A. To prevent the possible renaturation of
PhoA, which may affect its degree of aggregation, the same buffer
containing 100 mM iodoacetamide was used in parallel
experiments, but no differences were observed.
Degradation of PhoA(
To identify the responsible proteolytic pathway, we constructed by
P1vir transduction mutant strains in which genes coding for
different ATP-dependent proteases are disrupted (Table
I). As shown in Fig. 1, inactivation of
the lon gene reproducibly increased the half-life of
PhoA( DnaJ Is Required for PhoA Degradation by Proteases La and
ClpAP--
Previous studies demonstrated that DnaK and its cofactors
are required for the degradation of PhoA61, and that the rate of degradation correlates with the extent of association between the
substrate and DnaK (6). Because DnaJ has been shown also to bind
directly to certain unfolded proteins (18-21), we investigated the
possible role of DnaJ in the breakdown of
PhoA( DnaJ Influences the Association of PhoA with the
Proteases--
One possible function of the chaperone could be to
facilitate the binding of the substrate to protease La and ClpAP. We
therefore measured the amount of these proteases associated with PhoA
by immunoprecipitating PhoA from soluble cell extracts with an
anti-PhoA antibody, followed by quantitative Western blot analysis of
the precipitates with antibodies against La or ClpA. (The ClpA subunit was assayed in this experiment, because the anti-ClpA was more specific
than the anti-ClpP antibody we used.) Cell lysates were prepared from
wild-type and dnaJ ts-mutant cells, which had been shifted
to 37 °C for 20 min. In extracts of wild-type cells, some La and
ClpA could be coimmunoprecipitated with PhoA, as shown in Fig.
3, but none was detected in control
immunoprecipitations from cells lacking
PhoA(
Further evidence that the dnaJ mutant reduces the
association of PhoA and the ClpAP protease was obtained when the
converse experiment was performed, in which ClpA was immunoprecipitated from extracts of wild-type and mutant cells at 37 °C with an
anti-ClpA antibody. When the amount of PhoA present in the precipitates was analyzed by Western blot with an anti-PhoA antibody (not shown), much less PhoA was present in the dnaJ mutant than in
controls. Furthermore, in the dnaK756 mutant, where PhoA
degradation was accelerated (Ref. 6, and see below), much more
ClpA was coprecipitated with PhoA than in wild-type cells (not shown).
Additional experiments indicated that the chaperone, although affecting
protease-substrate interactions, is not necessary for the formation or
maintenance of active proteases, because the specific peptide
substrates of protease La, Suc-Ala-Ala-Phe-4-methoxy-2-naphtylamide,
and ClpAP, Suc-Leu-Tyr-MNA, were cleaved at similar rates in the
wild-type and dnaJ mutants growing at 37 °C. Thus,
DnaJ's ability to support PhoA breakdown correlates with its ability
to enhance the association of this substrate with these
ATP-dependent proteases.
Amount of PhoA Associated with DnaJ Also Correlates with Its
Degradation Rate--
Because the association of a protein with DnaK
(or DnaJ) is likely to be quite brief during successful protein folding
but much longer with a mutant protein that is unable to fold correctly, we proposed that the prolonged association of PhoA61 with DnaK might be
the critical factor leading to its degradation (7). Accordingly, the
extent of DnaK binding to PhoA was found to correlate with its rate of
degradation (6). Because DnaJ has also been shown to form complexes
with many unfolded proteins and with DnaK (20), a substrate that
dissociates poorly from DnaK might also be found in a prolonged
association with DnaJ. To test if DnaJ also associates with PhoA during
its degradation, we first immunoprecipitated PhoA from soluble cell
extracts using an anti-PhoA antibody and then performed quantitative
Western blot on the immunoprecipitates with an antibody against DnaJ
followed by detection with 125I-protein A. As shown in Fig.
4, very little DnaJ was found to coimmunoprecipitate with soluble PhoA under conditions where PhoA was
relatively stable, either at 30 °C in the wild-type or at 37 °C
in the dnaJ mutant, even though Western blots of extracts from these cells, using the antibody against DnaJ, demonstrated that
they contained similar amounts of this chaperone (data not shown). In
contrast, in the wild-type at 37 °C, where PhoA is rapidly degraded,
appreciable amounts of DnaJ were found to be associated with PhoA.
To further test whether the extent of the association with DnaJ
correlated with PhoA degradation rates, we constructed an isogenic
dnaK756 mutant (HHK2) and measured the rate of
PhoA(
It should be noted that, in our previous studies with PhoA61, which had
clearly indicated that rapid degradation also correlated with the
extent of DnaK binding, no DnaJ was found to coprecipitate under the
conditions used. However, in those experiments, we had included Triton
X-100 in the immunoprecipitation buffer (6). In the present studies,
DnaJ was detected in complexes with
PhoA( If DnaJ Is Inactivated, Some PhoA Aggregates--
Unfolded
proteins tend to aggregate in the absence of molecular chaperones
(22-24), and it has been proposed that the role of DnaJ and DnaK in
protein breakdown is primarily to help maintain the substrate in a
soluble form that allows proteolysis to occur (25). To determine
whether the DnaJ requirement for PhoA( PhoA Degradation and Solubility Do Not Require ClpB--
Recently,
the DnaK/DnaJ/GrpE system was shown to function together with
another molecular chaperone, ClpB, in disaggregation and refolding of
protein aggregates (23). Similarly in yeast, the homologous chaperones,
Hsp70 and Ydj-1, have been shown to function with the ClpB homolog,
Hsp104, in solubilizing protein inclusions (35). These observations
raised the possibility that DnaJ and DnaK promote
PhoA( Proper Folding of PhoA in a trxB Mutant Retards Its
Degradation--
The rapid hydrolysis of
PhoA( Role of DnaJ in a trxB Mutant--
To examine whether DnaJ is
needed for either the folding of PhoA or for its residual degradation
in the trxB strain, a trxB-dnaJ mutant was
constructed by transduction of a dnaJ259 mutation into WP552
(HHXJ6, Table I). The degradation of PhoA seen in the trxB cells was completely blocked at 37 °C in the trxB-dnaJ
double mutant (Fig. 7A). Furthermore, assay of alkaline
phosphatase activity in induced cells demonstrated ~3 times more
active enzyme in the trxB-dnaJ strain than in the
trxB parent (Fig. 7B). The prevention of
degradation of PhoA in the chaperone mutant thus led to its accumulation in an active form. In other words, proper disulfide bond
formation and folding of PhoA in the trxB cytosol do not require DnaJ.
We then examined whether the PhoA protein was present in soluble or
insoluble form in the trxB or trxB-dnaJ strain,
where a fraction of PhoA had enzymatic activity (Fig. 7B).
In both strains, the PhoA protein was detected almost exclusively in
the soluble fraction (Fig. 8). It is
interesting that almost none was found in aggregates (i.e.
15,000 × g pellets) in the trxB-dnaJ double mutant, in contrast to the dnaJ mutant, where a large
fraction was particulate (Fig. 8). Therefore, in an oxidizing
environment, where PhoA can form disulfide bridges, both native and
non-native, DnaJ is apparently not necessary for the folding process or
for maintaining it in a soluble form, even though this chaperone
remains absolutely essential for PhoA degradation.
The present studies indicate that the molecular chaperone, DnaJ,
plays multiple roles in determining the fate in the cytosol of a
nonsecreted alkaline phosphatase molecule,
PhoA( Previous findings in E. coli (6, 38) and yeast mitochondria
(11) have implicated the DnaK/DnaJ/GrpE chaperones in the rapid
breakdown of certain abnormal proteins by ATP-dependent proteases. Both the DnaK and DnaJ families of chaperones are known to
bind selectively to unfolded proteins (9, 15-17), and in this way,
they may promote the degradative process by facilitating the
recognition of unfolded proteins as substrates by the proteases. Alternatively, by maintaining the substrates in an unfolded
conformation, these chaperones may function as cofactors that
facilitate digestion by the proteases. Studies with another nonsecreted
variant, PhoA61, which contains a point mutation in the signal
sequence, had suggested a kinetic partitioning model for chaperone
function, in which a prolonged association of the substrate with DnaK
would favor proteolytic attack and help distinguish appropriate
substrates for degradation from normal cell constituents (7).
Successful folding of a protein, as occurs normally for most proteins,
would involve only a transient association with the chaperones, whereas an extended association, as may occur with a highly abnormal mutant protein or an irreversibly damaged polypeptide, would favor its proteolytic digestion.
The present finding that the association of PhoA with DnaJ correlates
with its rapid degradation is consistent with this proposed mechanism.
At 30 °C, where PhoA was relatively stable, or at 37 °C in the
dnaJ259 mutant, where PhoA degradation was blocked, much
less soluble PhoA was associated with DnaJ than in the wild-type at
37 °C. By contrast, in a strain carrying a nondissociating DnaK
mutation (dnaK756), where the amount of DnaJ complexed with PhoA was at least 5-fold higher than in the wild-type, PhoA degradation was enhanced at least 2-fold. In our prior studies, we had observed that the extent of DnaK association with PhoA61 (in dnaK756
and dnaK deletion strains) also correlated with the rate of
its hydrolysis (6). (Those earlier studies, in contrast with the
present ones, failed to demonstrate an association of PhoA with DnaJ
because of the use of the detergent Triton X-100 in the buffers.) Thus, these findings together strongly suggest that prolonged association of
this unfolded protein with DnaJ and DnaK facilitates its hydrolysis. However, these coimmunoprecipitation data only demonstrate correlations with the amount of PhoA present in complexes with the chaperones; the
data do not distinguish whether rapid degradation may in fact be due to
the presence of more molecules of the chaperone in complexes with the
substrate or to a more prolonged or a tighter association of the
substrate with the same number of chaperone molecules.
Although not directly demonstrated in these experiments, it seems very
likely that DnaJ and DnaK are present together in the same complexes
with PhoA, as was found for certain other denatured proteins (16, 17,
20, 21). For example, in the dnaK756 mutant, which is
defective in ATP-dependent dissociation from PhoA, more
PhoA was associated with DnaJ than in the wild-type. Also, the
dnaJ259 mutation, which is located in the highly conserved "J" domain that interacts with DnaK and thus causes a defective association with DnaK (39) showed a dramatically reduced association with PhoA. This in vivo failure of dnaJ259 to
bind to PhoA is noteworthy, because no deficiency had been observed
previously in its binding to model substrates in vitro. It
is not clear if DnaJ and DnaK influence degradation rates independently
or if one chaperone acts by influencing binding of the other as
suggested by the results with the dnaK756 mutant. Such
mechanistic questions will be best analyzed with pure components
in vitro.
The present results, although indicating a requirement of DnaJ for
proteolysis, also support the view that one function of this chaperone
in cells is to prevent PhoA aggregation into insoluble inclusions,
because 30-50% of the newly synthesized PhoA accumulated in rapidly
sedimenting fractions in the dnaJ mutant. A role for DnaJ in
maintaining denatured proteins in a soluble form has been established
in several studies (28, 40), especially in concert with DnaK (23, 24)
and also ClpB (23). In mitochondria, the rapid breakdown of two fusion
proteins by the Pim1 protease (a homolog of protease La) required the
mitochondrial DnaJ homolog Mdj-1, and in its absence, some of these
proteins accumulated as insoluble aggregates (11). Similarly, in
E. coli, the rapidly degraded transcriptional activator RcsA
was stabilized in a dnaJ mutant, and a large fraction of the
RcsA was found in insoluble aggregates (26). It was therefore proposed
that the primary role of DnaJ, and by extension DnaK and GrpE, in
protein breakdown is to maintain the substrate soluble and therefore
susceptible to the protease (25). However, there is no evidence that
maintenance of these molecules in a soluble form is actually essential
for proteolysis. Although it is likely that proteolytic attack on aggregated substrates may be slower and less efficient than degradation of soluble proteins (at comparable concentrations), the automatic assumption (25) that polypeptides in particulate fractions cannot be
digested by soluble proteases seems unwarranted. Indeed, it has long
been known that denatured proteins, once in large inclusion bodies, can
still be rapidly hydrolyzed to amino acids (41, 42) and that protease
La can digest insoluble, membrane-associated proteins (43).
Rapid digestion of such aggregated species may also require the
involvement of molecular chaperones to promote substrate
solubilization, and for this reason, we investigated the possible
involvement in PhoA breakdown of ClpB, which can function with
DnaK/DnaJ/GrpE to solubilize aggregated proteins (23). ClpB,
unlike DnaK and DnaJ, was not necessary for proteolysis. However, the
clpB strain did consistently show a small reduction in PhoA
breakdown, perhaps because a minor portion of the substrate was
particulate and may require this chaperone for solubilization prior to
DnaJ-dependent degradation.
Although earlier studies (26), like the present ones, have demonstrated
a tendency of the stabilized abnormal proteins to aggregate, they did
not show (but assumed) that aggregation is the cause of the
stabilization. On the contrary, the present findings clearly indicate
an important additional function of chaperones in proteolysis, because
in the dnaJ mutant, degradation was abolished completely,
although at least half of the nondegraded PhoA remained soluble at
100,000 × g. Perhaps the best evidence for a role of DnaJ in facilitating the degradative process came from the
trxB strains, where the dnaJ mutant completely
blocked PhoA degradation, even though none of the substrate was aggregated.
In these trxB mutants, where a large fraction of the PhoA
was able to fold into the active conformation, the inactivation of DnaJ
led to a 3-fold increase in PhoA enzymatic activity. This increase in
activity was most likely due to the increase in the amount of soluble
PhoA that resulted from blocking its degradation. In other words, by
promoting PhoA degradation, DnaJ appears to suppress the folding
process. Such a role for DnaJ is supported by the finding that
overexpression of DnaK and DnaJ reduced the cytosolic accumulation of a
cloned human polypeptide (SPARC), presumably by increasing its
degradation, and it also reduced the yield of SPARC with correct
disulfide bond formation in a trxB mutant (44).
Interestingly, DnaJ itself has been reported to possess disulfide
oxidoreductase activity in vitro (45), and it is conceivable
that this activity may help maintain certain proteins, such as PhoA, in
an unfolded conformation in vivo (e.g. in a
trxB cell).
These findings suggest that an essential function of DnaJ and DnaK in
proteolysis is to facilitate the binding of substrates to the
ATP-dependent proteases, ClpAP or La, or to stabilize these enzyme-substrate complexes once they are formed. The chaperones may
promote the recognition of the partially unfolded protein by
maintaining certain domains in a conformation that is particularly susceptible to association with the proteases. The critical finding here is that, when DnaJ was defective, very little La or ClpAP appeared
to be associated with PhoA. These results indicate for the first time a
requirement for DnaJ for the formation of degradative complexes between
a substrate and the proteases that digest it. An analogous role of a
DnaJ homolog in targeting a substrate to proteolysis has been proposed
in yeast cytosol, based on the finding that binding of the DnaJ homolog
Ydj1 was necessary for ubiquitin conjugation to a rapidly degraded
protein (12). An additional example of Ydj1 maintaining a substrate in
a conformation that allows enzymatic attack is in the ubiquitination of
the Cln3 cyclin. In this case, Ydj1 binding to Cln3 was required for
Cln3 phosphorylation by p34Cdc28, which in turn triggers
ubiquitin-dependent proteolysis (46). Other chaperone
systems have also been implicated in "presenting" substrates for
proteolysis or helping maintain them in conformations that allow
proteolytic attack; for example, the rapid degradation of the fusion
protein, CRAG, in E. coli requires a specific association between the substrate GroEL and Trigger Factor (TF) (31, 47, 48).
It is very unlikely that the requirement for DnaJ in proteolysis is for
the folding, assembly, or activation of these two ATP-dependent proteases. Inactivation of these enzymes
seems unlikely in these experiments, because the cells were grown at
30 °C and switched to 37 °C for only 20 min, which was sufficient
to prevent complex formation between the protease and the substrate. In
addition, hydrolysis of specific fluorogenic peptide substrates by
these proteases occurred at similar rates in extracts of the wild-type and dnaJ mutant strains, and complex formation between ClpA
and ClpP occurred whether or not DnaJ was functional. Finally, Jubete et al. (26) have noted that inactivation of DnaJ stabilized some protein substrates of protease La, but not others. Therefore, the
chaperone must instead be necessary for substrate recognition or rapid digestion.
An important feature of the degradation of PhoA shown here is that this
process is catalyzed by two very different proteases, La and ClpAP. The
two ATP-dependent proteases contribute about equally
in vivo, and each can degrade the substrate apparently without the involvement of the other. Degradation by both proteases is
likely to occur in a highly processive fashion, because no intermediate
fragments of PhoA were observed; also with each, relatively stable
substrate-protease complexes could be captured by
coimmunoprecipitation. Previous findings with PhoA61 (6) and CRAG (31)
had raised the possibility of a specific collaboration between the
DnaK/DnaJ/GrpE chaperone system and protease La, and between the
GroEL/GroES/TF chaperones and the ClpP protease. The present findings,
however, indicate otherwise, that DnaJ somehow can facilitate the
interaction of PhoA with either protease La or ClpAP. Also, in related
studies, we have found that GroEL/GroES/TF can promote degradation of
other substrates by La (48). Thus, the specific chaperones required for
proteolysis seem to be dictated by the nature of the substrate, and not
by the protease that is involved.
A fundamental unresolved issue is what feature(s) of the nonsecreted
PhoA are recognized by DnaJ and/or DnaK; it is also unclear which
chaperone binds first. Because PhoA(2-22) fails to fold in the cytosol and
is rapidly degraded at 37 °C. We show that
PhoA
2-22 is degraded by two
ATP-dependent proteases, La (Lon) and ClpAP, and breakdown
by both is blocked in a dnaJ259-ts mutant at 37 °C. Both
proteases could be immunoprecipitated with PhoA, but to a much lesser
extent in the dnaJ mutant. Therefore, DnaJ appears to
promote formation of protease-substrate complexes. DnaJ could be
coimmunoprecipitated with PhoA, and the extent of this association
directly correlated with its rate of degradation. Although PhoA was not
degraded when DnaJ was inactivated, 50% or more of the PhoA remained
soluble. PhoA breakdown and solubility did not require ClpB. PhoA
degradation was reduced in a thioredoxin-reductase mutant
(trxB), which allowed PhoA
2-22
to fold into an active form in the cytosol. Introduction of the
dnaJ mutation into trxB cells further
stabilized PhoA, increased enzyme activity, and left PhoA completely
soluble. Thus, DnaJ, although not necessary for folding (or preventing
PhoA aggregation), is required for PhoA degradation and must play an
active role in this process beyond maintaining the substrate in a
soluble form.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-22) (27). The
present studies have focused on the role(s) of DnaJ, because this
chaperone has been suggested to bind directly to certain denatured
proteins and favor DnaK binding to the substrate (20) and also to play a crucial role in preventing aggregation of abnormal proteins (28). In
the reducing environment of E. coli cytosol, the normally periplasmic PhoA cannot form the disulfide bonds necessary for the
protein to acquire an active conformation (5). However, in a mutant
strain lacking thioredoxin reductase (trxB), the cytosol has
less reducing potential, and some PhoA(
2-22)
has been shown to fold into an active enzyme (29). We have therefore
also used the trxB strain to test whether the rapid
degradation of PhoA is in fact due to its failure to achieve the native
conformation and to determine what role DnaJ might play when folding
and degradation of this protein can both occur.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 Prime, Inc. (Boulder, CO) and
purified by adsorption to extracts of a strain lacking the
PhoA gene. The polyclonal anti-DnaJ antibody was a kind gift
of Dr. R. McMacken (Johns Hopkins University), and anti-La, anti-ClpA,
and anti-ClpP antibodies were a kind gift of Dr. C. H. Chung
(Seoul National University, Korea). Protein A-Trisacryl beads were
obtained from Pierce, and 125I-protein A was from ICN. All
other reagents were purchased from Sigma and were of the highest purity available.
-D-galactopyranoside (IPTG)1 for 20 min to induce
PhoA expression. Degradation was assayed at 37 °C after blocking
protein synthesis with the addition of a mixture of antibiotics,
including chloramphenicol (100 µg/ml) and rifampicin (300 µg/ml) at
t = 0. Aliquots of cell culture were taken in
duplicates, immediately and at the indicated times thereafter. Cell
proteins were precipitated with 10% trichloroacetic acid, washed with
acetone, resuspended in SDS-PAGE sample buffer, and boiled. Samples
were resolved on 12% SDS-PAGE, transferred onto a nitrocellulose
membrane, and blotted with an anti-PhoA antibody and
125I-protein A. Quantification of PhoA band intensity was
performed with a PhosphorImager, and the duplicates agreed to within
5%.
2-22). Cells were then washed and lysed
by SDS/CHCl3, and PhoA activity was assayed at 28 °C by
the addition of a chromogenic substrate p-nitrophenyl phosphate and detected at A420. Assays
were performed in duplicates, and the results varied by less than
2%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-22) Involves Multiple
ATP-dependent Proteases--
Without its signal sequence,
E. coli alkaline phosphatase (PhoA) is not secreted into the
periplasm, and in the reducing environment of the cytoplasm, it fails
to form the disulfide bonds necessary for its native conformation and
enzymatic activity (5). To follow the fate of this protein, we used a
strain (WP551) that carries on a plasmid a wild-type alkaline
phosphatase gene that lacks a signal sequence, under the control of the
inducible tac promoter (PhoA
2-22)
(32). After induction of PhoA for 20 min, protein synthesis was
blocked, and the loss of PhoA antigen with time was measured. The newly
synthesized PhoA(
2-22) was rapidly degraded. Its rate of degradation increased sharply with the growth temperature, and the half-life of PhoA ranged from more than 30 min at
30 °C (not shown) to about 10 min at 37 °C (Fig.
1). These findings resemble closely our
earlier observations (6) with another nonsecreted inactive variant of
PhoA (PhoA61), which has a point mutation in its signal sequence. It
had previously been hypothesized that this altered signal sequence
might serve as the recognition element for the protease or a molecular
chaperone cofactor. Because a similar temperature-dependent
degradation occurred with the two nonsecreted variants, the cytoplasmic
degradative system does not recognize the signal sequence, and the
half-life of PhoA is apparently determined by the conformation of the
PhoA polypeptide itself.
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Fig. 1.
Rapid degradation of
PhoA( 2-22) involves both
ATP-dependent proteases La and ClpAP. Wild-type cells
and isogenic strains lacking functional lon,
clpA, clpP, or both lon and
clpP genes were grown at 30 °C to mid-logarithmic phase.
The cells were then shifted to 37 °C in the presence of IPTG for 20 min to induce PhoA expression. Degradation at 37 °C was measured
after stopping protein synthesis by the addition of a mixture of
antibiotics at t = 0. Aliquots of the culture were
taken at the indicated times, cell proteins were precipitated with
trichloroacetic acid, and the precipitates were resolved on SDS-PAGE.
The amount of PhoA remaining was measured by Western blotting with an
anti-PhoA antibody and 125I-protein A. Quantitation of the
PhoA band intensity was performed with a PhosphorImager. Similar
results were obtained in several independent experiments.
2-22) from 10 to 20 min at 37 °C.
This 2-fold stabilization in the lon mutant is similar to that observed with PhoA61 in lon- cells (6), but the
protease responsible for the residual degradation had not previously
been identified. To test if the remaining degradation was by another ATP-dependent protease, we measured the half-life of
PhoA(
2-22) in a clpP mutant. Its
rate of degradation was also about 2-fold slower in this mutant than in
the wild-type (Fig. 1). Because ClpP can function in complex with
either the ClpA or ClpX ATPase (33, 34), we also tested if inactivation
of one of these ATPases affected PhoA degradation. In a clpA
mutant, the half-life of PhoA(
2-22) increased
to a very similar extent as in the clpP mutant
(t1/2 ~ 20 min, Fig. 1). Thus, protease La (Lon)
and the ClpAP protease (also called Ti) are both involved in the
degradation of this substrate. Accordingly, in a lon-clpP
double mutant, PhoA(
2-22) was dramatically
stabilized (t1/2 > 2 h) (Fig. 1). Therefore, both La and ClpAP proteases contribute approximately equally to the
very rapid breakdown of PhoA in the cytosol, but each can function by
itself in this process.
List of strains used in this study
2-22). A dnaJ259-ts mutation
was transduced into WP551. The resulting temperature-sensitive strain,
HHJ1 (Table I), grew well at 30 °C or 37 °C, but did not grow at
all at 43 °C (data not shown). These cells were grown at 30 °C to
mid-logarithmic phase, shifted to 37 °C in the presence of IPTG for
20 min to induce PhoA expression, and then the degradation of PhoA
(
2-22) was measured. In these cells, this
degradative process was completely blocked at 37 °C (Fig.
2), even though cell growth still
occurred at this temperature. Because the requirement for DnaJ for PhoA
degradation is more stringent than that for cell growth, DnaJ must be
serving some highly specific, essential role in this degradative
process. Moreover, because PhoA(
2-22) was
completely stabilized in the dnaJ mutant (Fig. 2), this
chaperone is required for the degradation of PhoA by both proteases La
and ClpAP, each of which accounts for about half the degradation seen (Fig. 1).
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Fig. 2.
DnaJ function is required for the degradation
of PhoA( 2-22). Degradation at
37 °C was assayed in isogenic wild-type (wt) and mutant
strains as in Fig. 1. The effect of the dnaJ259-ts mutation
was compared with that of a dnaK756 mutation, which had been
shown to prevent PhoA release from DnaK in response to ATP (6). Similar
results were obtained in at least two additional experiments.
2-22) or when a mock immunoprecipitation was carried out with only protein A-beads or preimmune serum (not shown). By contrast, after the inactivation of DnaJ at 37 °C, the
amount of ClpA or La that coprecipitated with soluble PhoA was much
lower in the mutant than in the wild-type. This inactivation of DnaJ
did not affect the association between ClpA and ClpP, because the
amount of ClpP that coprecipitated with an anti-ClpA antibody was
similar in the wild-type and in the dnaJ mutant (data not
shown).
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Fig. 3.
DnaJ promotes the association of
PhoA( 2-22) with proteases La and
ClpAP. The wt and dnaJ259 cells were grown
at 30 °C, and PhoA expression was induced at 37 °C. PhoA was
immunoprecipitated from the soluble cell lysates with an anti-PhoA
antibody. The precipitates were resolved by SDS-PAGE and transferred
onto a nitrocellulose membrane for Western blot analysis. The amounts
of protease La and ClpA that coprecipitated with PhoA were assayed with
an anti-La or anti-ClpA antibody followed by 125I-protein
A. Similar results were obtained in several additional experiments. No
protease La or ClpAP was detected in similar immunoprecipitations from
cells lacking PhoA(
2-22) or in mock
immunoprecipitations using preimmune serum.
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Fig. 4.
DnaJ can be coimmunoprecipitated with
PhoA( 2-22), and this association correlates
with the rate of PhoA degradation. Cells were initially grown at
30 °C, and PhoA expression induced in the wild-type (wt)
at 30 °C (where PhoA is relatively stable) and in the wt,
dnaJ259, and dnaK756 strains after transfer to
37 °C. After precipitation from the soluble cell extracts with an
anti-PhoA antibody, the amount of precipitated PhoA was confirmed by
Western blot, and the presence of DnaJ that coprecipitated with PhoA
was detected with an anti-DnaJ antibody. The amounts of PhoA from the
different strains were normalized, and the amount of DnaJ that
coprecipitated was expressed relative to that in the wt at
37 °C. The intensities of PhoA and DnaJ bands in the wt
were set as 100. Quantitation of band intensity was performed with a
PhosphorImager. Similar results were obtained in at least two
additional experiments.
2-22) breakdown as well as the amount of
DnaJ in complex with this substrate. This mutation was previously shown
to cause a failure of PhoA61 (and other polypeptides) to be released
from DnaK, even in the presence of ATP (6). The rate of degradation of
PhoA(
2-22) clearly increased in this mutant,
from a t1/2 of about 10 min to one of less than 5 min at 37 °C (Fig. 2), and the relative amount of DnaJ in complex
with soluble PhoA (
2-22) was almost 5-fold
higher than in wild-type (Fig. 4). In control (mock)
immunoprecipitations from all three strains, using only protein
A-beads, no DnaJ was detected (data not shown). Thus, when PhoA was
degraded faster, more of it was present in complexes with DnaJ (as well
as with DnaK (6)). These findings with the dnaK756 and the
dnaJ mutants suggest a link between enhanced or reduced
chaperone binding and changes in degradative rates.
2-22), because Triton X-100 was removed
from the buffer. Also, the amount of DnaJ that coprecipitated with PhoA
was not affected by the addition of Mg-ATP or EDTA, in contrast to
DnaK, which dissociated from PhoA upon ATP addition (data not shown).
2-22) degradation was to prevent its becoming insoluble, we examined whether
the nondegraded protein was soluble or in particulate form. After
shifting the cells grown at 30 to 37 °C, PhoA expression was induced
for 20 min. Cells were then disrupted by sonication and subjected first
to centrifugation at 15,000 × g for 15 min to remove
large insoluble protein aggregates, and subsequently the supernatant
was ultracentrifuged at 100,000 × g for 1 h.
These pellets were resuspended in the original buffer volumes, and the amount of PhoA in each fraction was measured by quantitative Western blot. In wild-type (WP551) cells, all of the PhoA protein was found in
the soluble fractions (i.e. 100,000 × g
supernatant) (Fig. 5). In the
lon-ClpP double mutant, where degradation of PhoA was
completely abolished (Fig. 1), it remained almost entirely soluble even
after the ultracentrifugation step (Fig. 5). However, in the
dnaJ mutant, where PhoA was also not degraded, ~50% of the protein was recovered in the 15,000 × g pellet,
and the remainder was soluble. The degree of PhoA aggregation was also
determined after a 1-h incubation at 37 °C, and the amount of PhoA
in the 10,000 × g or 100,000 × g
pellets did not increase further during this period (data not shown).
Thus, DnaJ clearly helps to maintain some of the nondegraded PhoA in a
soluble form. However, even without functional DnaJ, half of these
molecules remained soluble after centrifugation at 100,000 × g (Fig. 5). Because degradation of both the soluble and
insoluble PhoA was completely blocked under these conditions (Fig. 2),
DnaJ must play a direct role in the degradation of soluble PhoA, beyond
any additional role in preventing aggregation of some of the
nondegraded molecules.
View larger version (36K):
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Fig. 5.
When DnaJ is inactivated, some PhoA becomes
insoluble. Cells were grown at 30 °C, shifted to 37 °C in
the presence of IPTG for 20 min to induce PhoA expression, then
disrupted by sonication to yield total lysates (T). The
subcellular distribution of PhoA was compared in wild-type cells and in
the isogenic dnaJ259 and lon-clpP strains where
PhoA is stable. Centrifugation of T at 15,000 × g for 15 min produced the low speed pellet
(PL) and supernatant (SL).
SL was then subjected to ultracentrifugation at
100,000 × g for 1 h to give the high speed pellet
(PH) and supernatant (SH). The
fractions were resolved on SDS-PAGE, and the amount of PhoA present in
each was detected by Western blot with an anti-PhoA antibody and
125I-protein A. Identical results were obtained in several
independent experiments.
2-22) degradation by functioning
together with ClpB to solubilize aggregated PhoA molecules. To test
whether ClpB is also essential in PhoA degradation, a clpB
deletion (strain kindly provided by Dr. T. Baker, MIT) was introduced
into WP551 background by P1 transduction. Using this strain, we tested
whether ClpB was a cofactor in PhoA degradation. Although its
degradation was consistently reduced to a slight extent in the
clpB mutant (Fig.
6A), this reduction was much
smaller than that seen upon DnaJ inactivation or upon loss of DnaK,
which caused a complete stabilization of PhoA. Thus, at 37 °C, ClpB
is not an essential cofactor in DnaJ/K-dependent
degradation of PhoA. Moreover, in the clpB mutant at
37 °C, nearly all the PhoA remained soluble (Fig. 6B), in
contrast to the dnaJ mutant, where about 50% of the protein
was insoluble. Thus, ClpB also is not important to maintain PhoA
soluble at this temperature.
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Fig. 6.
ClpB function is not essential for the
degradation of PhoA( 2-22) at 37 °C
or for keeping it soluble. A, inactivation of ClpB
slightly reduces the rate of PhoA(
2-22)
degradation. Degradation at 37 °C was assayed in isogenic wild-type
and mutant strains as in Fig. 1. The effect of the clpB
mutation was compared with that of dnaJ259 mutation. Similar
results were obtained in several experiments. B, when ClpB
is inactivated, PhoA remains soluble. The subcellular distribution of
PhoA was assayed as in Fig. 5.
2-22) at 37 °C results presumably
from its failure to form sulfhydryl bridges in the cytosol and to
achieve a stable conformation. The active form of PhoA (alkaline
phosphatase) contains two intra-chain disulfide bonds. Beckwith and
coworkers (29) have shown that cytosolic proteins are maintained in a
reduced state in part due to the function of thioredoxin reductase
(TrxB). In a trxB mutant, all the nonsecreted PhoA(
2-22) was able to form disulfide
bridges, and a substantial portion could fold into a native form in the
cytoplasm (29). Accordingly, although very little alkaline phosphatase
activity was observed in the wild-type parent (WP551) expressing PhoA
(
2-22), this activity was clearly evident in
the cytosol after a disrupted trxB gene was transduced into
the WP551 strain (WP552, Table I). We therefore tested whether this
formation of the proper disulfide bonds in PhoA also influenced its
degradation. As shown in Fig. 7A, the breakdown of PhoA was
30-40% slower in the trxB mutant. Thus, proper folding of
PhoA in the cytosol appears to reduce its degradation. This partial
inhibition of degradation may indicate that only a fraction of the
PhoA(
2-22) was able to fold into the active
conformation, and it alone may escape proteolysis (see below).
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Fig. 7.
Proper folding of PhoA in a trxB
mutant retards its degradation, and DnaJ is required for this
degradation but not for PhoA folding. The isogenic wt,
trxB, and trxB-dnaJ259 strains were grown and
PhoA induced at 37 °C as in Fig. 5. A, PhoA degradation
was assayed at 37 °C as in Fig. 2. B, alkaline
phosphatase activity in these strains was measured as described under
"Experimental Procedures." Similar results were obtained in three
separate experiments.
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Fig. 8.
PhoA is soluble in a trxB
mutant even without functional DnaJ. The strains were grown
and lysed, and PhoA was induced at 37 °C as in Fig. 5. Fractionation
of the cell lysates and measurement of PhoA content were carried out as
in Fig. 5 after centrifugation of the total lysate at 15,000 × g for 15 min: T, total lysate; S,
15,000 × g supernatant; P, 15,000 × g pellet. Identical results were obtained in at least two
additional experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-22). Normally, when a signal
peptide is present, DnaJ, together with DnaK and GrpE, promotes
translocation of this enzyme into the periplasm (36, 37). When export
is impossible, DnaJ, apparently with DnaK and GrpE (6), carries out
some function essential for rapid degradation of this unfolded protein
by the ATP-dependent proteases La (Lon) and ClpAP. Although
DnaJ was found to help maintain this protein in a soluble form (for
example, in the lon-ClpP mutant cell, where degradation is
not possible), DnaJ clearly must play additional role(s) in PhoA
degradation beyond preventing the substrate from forming insoluble
aggregates. When DnaJ was inactivated, at least half of the PhoA
molecules remained soluble at 100,000 × g, yet they
were not degraded. Furthermore, when a favorable redox environment was
provided (i.e. in the trxB mutant cells), in
which some PhoA can fold into its native form, DnaJ was still required
for its degradation, but was not necessary for the prevention of
aggregation or for the proper folding of the active enzyme.
2-22),
which lacks the entire signal sequence, was degraded rapidly in a
similar process as PhoA61, substrate recognition cannot be through
chaperone binding to the unfolded signal sequence. That mechanism was
initially an attractive one, because an uncleaved signal sequence would be a simple method for recognition of a protein that failed to be
successfully translocated into the periplasm (49). Presumably, exposed
hydrophobic domains in the PhoA polypeptide itself are recognized by
DnaJ or DnaK (14). Other important questions include the precise roles
of DnaJ and DnaK in allowing PhoA to interact with the proteases. It
remains to be determined, for example, if binding of these chaperones
leads to the exposure of the C-terminal region of PhoA, to which ClpA
may bind through the recently identified PDZ-like domain common
to the Clp family (50). Possibly, prolonged association of a protein
with both chaperones may be specifically recognized by the proteases,
or may maintain the polypeptide in a conformation long enough for the
proteases to bind or to stay bound to the substrate. In either case,
the molecular chaperones would appear to be functioning as enzymatic
cofactors of the ATP-dependent proteases, rather than
indirectly promoting proteolysis.
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ACKNOWLEDGEMENTS |
---|
We thank Aurora Scott and Jodi Frisbie for help in preparing this manuscript and Dr. W. Prinz for useful advice and stimulating discussions throughout the course of this study.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants GM46147 and GM51293 from the National Institutes of Health (to A.L.G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Schering-Plough Research Institute, Kenilworth,
NJ 07033.
§ Present address: Boston Biomedical Research Institute, Boston, MA 02114.
¶ To whom correspondence should be addressed: Tel.: 617-432-1855; Fax: 617-232-0173; E-mail: alfred_goldberg@hms.harvard.edu.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M002937200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IPTG, isopropyl-1-thio--D-galactopyranoside;
PAGE, polyacrylamide gel electrophoresis;
TF, Trigger Factor.
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