(Received for publication, July 19, 1994; and in revised form, November 9, 1994)
From the
The universally conserved DnaK and DnaJ molecular chaperone
proteins bind in a coordinate manner to protein substrates to prevent
aggregation, to disaggregate proteins, or to regulate proper protein
function. To further examine their synergistic mechanism of action, we
constructed and characterized two DnaJ deletion proteins. One has an
11-amino-acid internal deletion that spans amino acid residues
77-87 (DnaJ77-87) and the other amino acids
77-107 (DnaJ
77-107). The DnaJ
77- 87 mutant
protein, was normal in all respects analyzed. The DnaJ
77-107
mutant protein has its entire G/F (Gly/Phe) motif deleted. This motif
is found in most, but not all DnaJ family members. In vivo, DnaJ
77-107 supported bacteriophage
growth, albeit
at reduced levels, demonstrating that at least some protein function
was retained. However, DnaJ
77-107 did not exhibit other wild
type properties, such as proper down-regulation of the heat-shock
response, and had an overall poisoning effect of cell growth. The
purified DnaJ
77-107 protein was shown to physically interact
and stimulate DnaK's ATPase activity at wild type levels, unlike
the previously characterized DnaJ259 point mutant (DnaJH33Q). Moreover,
both DnaJ
77-107 and DnaJ259 bound to substrate proteins,
such as
, at similar affinities as
DnaJ
. However, DnaJ
77-107 was found to be
largely defective in activating the ATP-dependent substrate binding
mode of DnaK. In vivo, the ability of the mutant DnaJ proteins
to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind
,
in an ATP-dependent manner, and not with their ability to bind
. We conclude, that although the G/F motif of DnaJ
does not directly participate in the stimulation of DnaK's ATPase
activity, nevertheless, it is involved in an important manner in
modulating DnaK's substrate binding activity.
The DnaK (Hsp70 eukaryotic homolog), DnaJ (Hsp40 eukaryotic
homolog), and GrpE molecular chaperones participate in a variety of
biological process. Along with their central roles in protein folding
and transport (Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993),
they can modulate other aspects of protein function. For example, in
bacteriophage -DNA replication these chaperones catalyze the
disassembly of the DnaB helicase from the
P inhibitory protein in
a timely manner to allow DNA replication to initiate (Alfano and
McMacken, 1989; Zylicz et al., 1989). In addition, chaperones
may also regulate intramolecular protein interactions. For instance,
presumably through a conformationally induced change, DnaK can
``activate'' the DNA-binding properties of the tumor
suppressor p53 protein (Hupp et al., 1992).
Central to the
mechanism of action of this chaperone machine, DnaJ stimulates the
hydrolysis of DnaK-bound ATP (Liberek et al., 1991a). This
stimulation correlates with an increased affinity of DnaK for certain
substrates, i.e. (Liberek and
Georgopoulos, 1993), RepA (Wickner et al., 1991), luciferase
(Schröder et al., 1993), rhodanese (Langer et al., 1992), and
P (Osipiuk et al., 1993;
Zylicz, 1993). On the contrary, GrpE functions primarily as a
nucleotide exchange factor for DnaK (Liberek et al., 1991a).
In some instances, this function of GrpE has been shown to correlate
with substrate release from DnaK, i.e. rhodanese (Langer et al., 1992) and
P (Hoffmann et al., 1992;
Osipiuk et al., 1993). Jointly, DnaJ and GrpE stimulate
DnaK's ATPase activity
50-fold and likely facilitate the
recycling of these chaperone proteins (Liberek et al., 1991a).
Previously we have established that the DnaJ12-truncated protein,
containing only the amino-terminal 108 amino acids of DnaJ is necessary
and sufficient to (i) stimulate DnaK's ATPase activity, (ii)
regulate DnaK's conformational state in the presence of ATP,
(iii) activate DnaK to bind in the presence of ATP
and, (iv) elicit proper, albeit limited, chaperone function from DnaK
for
-DNA replication (Wall et al., 1994). (
)These conclusions were based primarily on the findings
that DnaJ12 possessed all these functions, while DnaJ259, which carries
a single His
Gln substitution at residue 33, lacked all of these
activities. Here, we address the function of the G/F motif of DnaJ,
which comprises amino acid residues 77-107.
Sequence analysis of over 30 DnaJ family members from prokaryotic and eukaryotic organisms suggests that the amino-terminal 108 amino acids of DnaJ consists of two well-conserved domains or modules. The first 76 amino acids constitute the most highly conserved region of DnaJ that has been used as the ``signature'' sequence to identify additional DnaJ family members, and is thus called the J-domain (Bork et al., 1992; Silver and Way, 1993; Wall et al., 1994). The next 31 amino acids are rich in the amino acids Gly and Phe and are therefore referred to as the G/F motif or module (Fig. 1). Since this region is rich in gly residues, it has been suggested that the G/F motif functions as a linker between the J-domains and the rest of the DnaJ protein (Silver and Way, 1993). However, an amino acid alignment of this motif reveals a considerable amount of sequence conservation (Fig. 1). In particular, there is a nearly universally conserved Asp-Ile-Phe (DIF) motif (in two cases Ile has been replaced by Val, a conservative substitution). Escherichia coli's DnaJ has two additional DIF repeats (Fig. 1). The existence of these highly conserved residues suggests that this DIF motif may be important for the structure/function of DnaJ. The alignment also reveals a certain amount of flexibility in the number of Gly residues. Interestingly, some DnaJ family members do not contain this motif, although most do (Bork et al., 1992), suggesting that the G/F motif is dispensable for some of DnaJ's functions. Moreover, the fact that the G/F motif is always found immediately downstream of the J-domain has led to the suggestion that it may play an auxiliary role in modulating DnaK's chaperone activity, synergistic with the J-domain (Wall et al., 1994).
Figure 1:
Alignment of the G/F motif present in
most DnaJ family members. Sequences homologous to E. coli DnaJ
were obtained using the TFastA search program (Pearson and Lipman,
1988) of the GenEMBL data base. Sequences were aligned using the
Genetics Computer Group (GCG) program PileUp (gap weight, 3.0; gap
length, 0.1). A consensus sequence was obtained with the Pretty program
(plurality, 7; threshold, 1.0). Residues that contributed toward the
consensus are capitalized, while residues that are identical
in more than 80% of the sequences are indicted as white letters with a black background. An abbreviation of the
organism's or proteins (yeast) name is given in the left margin.
The G/F residues, deleted in the DnaJ77-87 and
DnaJ
77-107 constructs, are indicated by black
bars.
To address what role the G/F motif plays in
DnaJ function, we constructed two DnaJ internal deletion mutants. The
first, DnaJ77-87, has the first 11 amino acids deleted, 9 of
which are Gly residues (Fig. 1). The highly conserved DI(V)F
residues are not deleted in this construct. In contrast,
DnaJ
77-107 has the entire 31 amino acid G/F motif deleted (Fig. 1, see ``Materials and Methods''). The genes
that code for these mutants are carried on the pTTQ19 plasmid (Amersham
Corp.), under the tight transcriptional control of the lacI
gene product, whose gene is simultaneously
carried on the plasmid. To test for functional activity, these plasmids
were transformed into E. coli strains that have been deleted
for the dnaJ gene. Interestingly, the DnaJ
77-87
mutant complemented all bacterial and bacteriophage
growth
defects associated with the dnaJ null mutants to near wild
type levels at either 30 or 42 °C. In contrast, the
DnaJ
77-107 mutant did not complement any of the bacterial
growth defects associated with the DnaJ null mutant. In fact, cells
harboring the DnaJ
77-107 allele grew considerably slower
than the isogenic bacterial strains that carried the parental vector
plasmid, suggesting that the slightly overproduced (
2-fold)
DnaJ
77-107 was somehow poisonous to cell growth, perhaps
through nonproductive interactions with DnaK (see below).
The
DnaJ77-107 protein was able to complement bacteriophage
growth, albeit much less efficiently (
50-fold lower
bacteriophage yield) than the DnaJ
protein in the same
background, demonstrating that at least some of its functions were
retained. Bacteriophage
grew to a similar extent at either 30 or
42 °C, showing that DnaJ
77-107 does not exhibit a
temperature-sensitive phenotype for
growth per se as
other dnaJ mutants do (Wall et al., 1994), but simply
works less efficiently at all temperatures compared to
DnaJ
. On the basis of these in vivo results,
we decided to biochemically characterize in detail the
DnaJ
77-107 mutant protein, not only because it exhibited
interesting phenotypes, but also because the DnaJ
77-87
protein appeared to have identical activities as DnaJ
.
Figure 2: Interactions of various DnaJ proteins with DnaK. A, titration of the ability of DnaJ proteins to stimulate DnaK's ATPase activity. The reaction mixtures contained ATP (200 µM), DnaK (0.41 µM), GrpE (0.90 µM), and varing amounts of DnaJ proteins, as described (Liberek et al., 1991a). B, ability of DnaJ proteins to bind DnaK. The indicated DnaJ proteins (0.5 µg/well) or BSA (100 µg/well) were added to microtiter plate wells and treated as described under ``Materials and Methods.'' Following a series of blocking and washing steps (see ``Materials and Methods''), various amounts of DnaK were added for 45 min at room temperature. Subsequently, the amount of bound DnaK protein was measured by ELISA using rabbit antiserum against DnaK (1:7,000 dilution) as described under ``Materials and Methods.''
Our
earlier work established that a His Gln substitution at amino
acid 33 (DnaJ259) resulted in a protein that was completely defective
in stimulating DnaK's ATPase activity (Wall et al.,
1994). However, this result did not distinguish between a possible
defect in the physical interaction between DnaK and DnaJ259, or
alternatively a potential catalytic defect in DnaJ259. To establish
what role the J-domain and G/F motifs play in DnaJ's physical
interaction with DnaK, we tested the ability of DnaJ259 and
DnaJ
77-107 mutant proteins to bind DnaK. To assay for this
protein-protein interaction we used a modified ELISA approach (see
``Materials and Methods''), since it was recently shown to be
a sensitive assay to monitor DnaK-DnaJ interactions. (
)Fig. 2B shows that in the presence of ATP
DnaJ
77-107 exhibits a high affinity toward DnaK, nearly
identical to that of wild type DnaJ. In contrast, DnaJ259 was severely
defective in its ability to bind DnaK (Fig. 2B). These
results are in good overall agreement with the ATPase stimulation data.
In addition, as judged by partial trypsin digestion analysis,
DnaJ
77-107 was found to regulate DnaK's conformational
state in an ATP-dependent reaction similar to DnaJ
(Wall et al., 1994; data not shown). Therefore, we
conclude that the J-domain plays the major role in determining the
biochemical affinity of DnaJ to DnaK.
Figure 3:
Ability of various DnaJ proteins to bind
and to activate DnaK to bind
. A, the indicated DnaJ proteins (0.5 µg/well) or BSA (100
µg/well) were incubated with various amounts of
as described under ``Materials and Methods.'' The
amount of bound
protein was measured by ELISA using
rabbit antiserum against
(1:2,000 dilution) as
described under ``Materials and Methods.'' B,
protein (0.5 µg/well) or BSA (100 µg/well)
were incubated with DnaK (80 ng), ATP (1 mM) and various
amounts of DnaJ proteins. The amount of DnaK bound to the
or BSA wells was estimated by ELISA using antiserum to DnaK
(1:7,000 dilution) as described under ``Materials and
Methods.''
In parallel experiments, we confirmed and
extended our earlier results, demonstrating that DnaJ259 was completely
defective in activating DnaK to bind in an
ATP-dependent reaction by a factor of at least 100. Interestingly,
DnaJ
77-107 was severely defective in its ability to activate
DnaK to bind
(Fig. 3B), although it
exhibited a weak, yet reproducible stimulatory activity. In analogous
experiments in which firefly luciferase was substituted for
, a protein known to interact with DnaK and DnaJ
(Schröder et al., 1993; Wall, 1994),
DnaJ
77-107 was again found to be defective in activating
DnaK to bind luciferase in an ATP-dependent reaction as compared to
DnaJ
(Wall, 1994).
In a second approach, glycerol
gradient sedimentation also showed that DnaJ77-107 was
unable to activate DnaK to bind
in the presence of
ATP (not shown). These results indicate that the stimulatory role DnaJ
plays in DnaK's ATPase activity is not sufficient by itself to
activate DnaK to bind to at least some of its substrates. Thus, we
conclude that the G/F motif plays an auxiliary, yet important, role in
activating DnaK to bind substrates that is clearly separable from the
stimulatory role that DnaJ plays in stimulating DnaK's ATPase
activity.
To test
whether or not the important biochemical interaction in vivo for heat-shock autoregulation was the DnaK- interaction, we tested the ability of the dnaJ12 and dnaJ
77-107 mutant alleles to down-regulate the
heat-shock response, as judged by the expression from a heat-shock
reporter gene fusion, groE::lacZ. Using isogenic bacteria we
found that the dnaJ12 allele could down-regulate the
overproduction of the heat-shock response caused by a dnaJ
null mutation, while dnaJ
77-107 could not (Fig. 4). As controls,
the parental vector alone or the dnaJ259-bearing plasmid could
not down-regulate the expression of the groE::lacZ reporter
gene, while an isogenic dnaJ
-containing
plasmid could (Fig. 4). Consistent with our other results, the dnaJ
77-87-containing plasmid was able to
down-regulate the expression of the groE::lacZ reporter gene (Fig. 4). These results are highly reproducible since they were
obtained with two independently transformed strains, and six
independent
-galactosidase assays gave similar results.
Figure 4:
Ability of various DnaJ proteins to down
regulate E. coli's heat-shock response. A dnaJ mutant strain, that contains the
heat-shock reporter gene ØpgroE-lacZ integrated into
the chromosome, was transformed with the indicated plasmid-borne dnaJ alleles or the plasmid vector. The
-galactosidase
activity (Miller units) of these isogenic bacteria was then determined
at 30 °C as described previously (Miller, 1992). The values given
are an average of six independent
determinations.
To
further substantiate these results, the same plasmids were transformed
into a dnaJ mutant strain, carrying a
different heat-shock reporter gene fusion lon::lacZ (Missiakas et al., 1993). Again, DnaJ12 was found to down-regulate
transcription from this heat-shock reporter fusion to a level similar
to that seen with isogenic dnaJ
bacteria,
while the dnaJ259- and dnaJ
77-107-bearing
isogenic plasmid constructs did not (data not shown). Together with our in vitro data, we conclude that it is the DnaJ-catalyzed
ability of DnaK to recognize and bind
that is
critical to the down-regulation of the heat-shock response in E.
coli and not the ability of DnaJ to bind
, as
has been previously suggested by others (Bukau, 1993). As previously
mentioned, the DnaJ protein levels produced from our expression plasmid
vectors are no more than 2-fold higher than those found for wild type
bacteria expressing DnaJ from a single copy gene, as judged by
quantitative Western analysis (not shown).
A central question in the protein chaperone field is how DnaK
and its eukaryotic counterparts bind and release substrates. Increasing
evidence indicates that in vivo substrate binding by DnaK is
often a coordinated process with DnaJ and ATP (Langer et al.,
1992; Liberek and Georgopoulos, 1993; Osipiuk et al., 1993;
Wickner et al., 1991; Zylicz, 1993). We have shown here and
elsewhere that the critical steps in this concerted process involve the
ability of DnaJ to stimulate ATP hydrolysis by DnaK, resulting in a
large conformational change in DnaK (Liberek et al., 1991a;
Wall et al., 1994). These steps are required for ATP-dependent
binding of DnaK to some of its substrates, since the DnaJ259 mutant
protein does not possess these activities and does not activate DnaK to
bind such substrates, while DnaJ12 has these activities and stimulates
DnaK to bind .
In this work we have
further investigated this important activation process by deleting the
conserved G/F motif (module), which is present in the DnaJ12 mutant,
while leaving the highly conserved J-domain and the rest of the DnaJ
protein intact. The resulting DnaJ77-107 mutant protein
contains a 31-amino-acid internal deletion substituted with a 6 amino
acid insertion. DnaJ
77-107 was found to stimulate
DnaK's ATPase activity like wild type DnaJ, but, surprisingly, it
does not activate DnaK to bind
. In this respect it
is interesting that Schmid et al.(1994) recently showed that
DnaK has a significantly faster on rate for substrates when bound to
ATP. However, this increased on rate for substrates is compensated by
an even faster off rate in the presence of ATP, thus the net result is that DnaK exhibits an approximately 10-fold lower
affinity toward substrates in the presence of ATP, as previously
established (Flynn et al., 1989; Liberek et al.,
1991b).
In agreement with biophysical and biochemical data, we interpret the process of ATP binding to DnaK as an ``activation'' step, in which the substrate binding domain/pocket of DnaK becomes more exposed to the solvent (Buchberger et al., 1994; Liberek et al., 1991b; Palleros et al., 1991; Schmid et al., 1994). Although substrates may bind faster in this conformation, they are also released at a fast rate (Schmid et al., 1994). In our model, the role of DnaJ is to both help deliver substrates to this substrate-binding pocket of DnaK and to stimulate pocket closure (i.e. conformational change), presumably through ATP hydrolysis. This regulated reaction ``locks'' DnaK into a conformation that stably binds substrates, i.e. slow off rate in the ADP form (Palleros et al., 1991; Schmid et al., 1994). This model is consistent with our findings that DnaJ ``triggers'' DnaK into the presumed ``closed'' conformation in the presence of ATP (Wall et al., 1994).
One interpretation of the failure of
DnaJ77-107 to activate DnaK to bind
is
that although DnaJ
77-107 stimulates ATP hydrolysis and DnaK
``closure,'' it fails to ``lock'' DnaK into a
conformation that stably binds
. This could be
explained mechanistically, for example, by a failure of
DnaJ
77-107 to regulate a possible phosphorylation or
oligomeric state of DnaK, which has been suggested to modulate
DnaK's affinity toward substrates (Schmid et al., 1994;
Sherman and Goldberg, 1993). (
)Alternatively, the
DnaJ
77-107 mutant protein could result in a conformational
perturbation such that it can no longer activate DnaK to bind
substrates. However, since DnaJ
77-107 can both stimulate
DnaK's ATPase activity and bind to substrates at wild type
efficiencies argues against DnaJ
77-107 being dramatically
misfolded. Along these lines, the failure of DnaJ
77-107 to
activate DnaK to bind substrates could result in a structural defect
such that it cannot simultaneously orchestrate substrate presentation
and ``activation'' to DnaK. In this interpretation, the G/F
module would serve as a linker or hinge between domains of DnaJ (Silver
and Way, 1993), thus allowing both domains to be properly oriented when
interacting with DnaK. In either interpretation, we conclude that the
G/F motif of DnaJ is serving an ``auxiliary,'' yet important
regulatory role in activating DnaK to bind substrates (see below).
Interestingly, some DnaJ family members do not contain the G/F motif
(Bork et al., 1992). For example Sec63, which is a Yeast
homolog and is a component of the protein transport machinery into the
endoplasmic reticulum, has a J-domain localized in the lumen of the
endoplasmic reticulum (reviewed in Sanders and Schekman, 1992). It is
thought that this domain interacts with the ER-localized BiP (Hsp70
homolog) in protein transport and folding (Feldheim et al.,
1992; Sanders and Schekman, 1992). We suspect that the coordinated
Sec63-BiP complex performs different biochemical functions than, for
instance, the binding of E. coli's DnaK/DnaJ to
or to the ``O-some'' complex in
DNA
replication (Zylicz, 1993). Most likely, the Sec63-BiP machinery
rapidly binds and releases unfolded/extended polypeptides
(Blond-Elguindi et al., 1993; Flynn et al., 1991) as
they are traversing the ER membrane, thus facilitating protein
transport and folding (Sanders and Schekman, 1992). Thus, in this
scheme Sec63 does not possess the G/F motif, simply because it does not
require the ``locking'' or ``stabilizing''
functions of DnaJ. In the examples of
and
P,
the DnaK/DnaJ machinery binds to ``native proteins'' (i.e. already in a biologically active form) and thus, most
likely, not present in an unfolded/extended state. Moreover, such
DnaK/DnaJ/substrate complexes may be longer lived, especially because
ATP hydrolysis ensures their stable association. Consistent with this,
Brodsky et al. (1993) found that Hsc70, Ssa1 (the DnaK
eukaryotic homologs), as well as E. coli's DnaK protein
could not substitute for BiP function in a reconstituted protein
transport system. Conversely, BiP did not substitute for the function
of cytosolic Hsc70, required for proper protein transport on the
cytosolic side of the ER membrane. Thus, although these DnaK-like
proteins are functioning in an overall similar manner to carry out
similar biological tasks, nevertheless, they are not interchangeable.
Recently, additional DnaJ and DnaK family members have been
discovered in E. coli (Kawula and Lelivelt, 1994; Seaton and
Vickery, 1994; Ueguchi et al., 1994; Yura et al.,
1992), bringing the total number of DnaJ family members to four and
DnaK family members to two. Interestingly, all other DnaJ family
members do not contain the G/F motif. This may reflect more specialized
roles for these putative DnaJ protein chaperones. Nevertheless, genetic
evidence indicates that at least one of these DnaJ family members can
functionally substitute for the ``classical'' DnaJ protein
(Ueguchi et al., 1994). For example, multicopy levels of the cbpA homolog gene restores bacterial growth at high
temperatures (Ueguchi et al., 1994), ()as well as
bacteriophage
growth to a dnaJ
strain
(Ueguchi et al., 1994). These results are also consistent with our
findings presented here, namely that multicopy levels of dnaJ
77-107 support
growth. The discovery of
the cbpA gene of E. coli helps explain the higher
basal level of
DNA replication activity found in a crude extract
system prepared from a dnaJ
strain compared to
a dnaJ259 strain (Wall et al., 1994).