(Received for publication, November 30, 1995)
From the
The DnaK and DnaJ heat shock proteins function as the primary
Hsp70 and Hsp40 homologues, respectively, of Escherichia coli.
Intensive studies of various Hsp70 and DnaJ-like proteins over the past
decade have led to the suggestion that interactions between specific
pairs of these two types of proteins permit them to serve as molecular
chaperones in a diverse array of protein metabolic events, including
protein folding, protein trafficking, and assembly and disassembly of
multisubunit protein complexes. To further our understanding of the
nature of Hsp70-DnaJ interactions, we have sought to define the minimal
sequence elements of DnaJ required for stimulation of the intrinsic
ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is
composed of three separate regions, a 9-kDa NH-terminal
domain, a 30-kDa COOH-terminal domain, and a protease-sensitive
glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids
that serves as a flexible linker between the two domains. The stable
9-kDa proteolytic fragment was identified as the highly conserved
J-region found in all DnaJ homologues. Using this structural
information as a guide, we constructed, expressed, purified, and
characterized several mutant DnaJ proteins that contained either
NH
-terminal or COOH-terminal deletions. At variance with
current models of DnaJ action, DnaJ1-75, a polypeptide containing
an intact J-region, was found to be incapable of stimulating ATP
hydrolysis by DnaK protein. We found, instead, that two sequence
elements of DnaJ, the J-region and the G/F-rich linker segment, are
each required for activation of DnaK-mediated ATP hydrolysis and for
minimal DnaJ function in the initiation of bacteriophage
DNA
replication. Further analysis indicated that maximal activation of ATP
hydrolysis by DnaK requires two independent but simultaneous
protein-protein interactions: (i) interaction of DnaK with the J-region
of DnaJ and (ii) binding of a peptide or polypeptide to the
polypeptide-binding site associated with the COOH-terminal domain of
DnaK. This dual signaling process required for activation of DnaK
function has mechanistic implications for those protein metabolic
events, such as polypeptide translocation into the endoplasmic
reticulum in eukaryotic cells, that are dependent on interactions
between Hsp70-like and DnaJ-like proteins.
The DnaJ, DnaK, and GrpE proteins of Escherichia coli were first identified via genetic studies of E. coli mutants that are incapable of supporting the replication of
bacteriophage DNA(1, 2, 3) . Later, it
was found that DnaJ, DnaK, and GrpE are all prominent bacterial heat
shock proteins, which comprise a set of about 30 proteins whose
expression is transiently induced when cells are grown at elevated
temperatures (reviewed in (4) and (5) ). In recent
years it has become apparent that eukaryotic cells contain families of
proteins that are homologous to each DnaJ and
DnaK(6, 7) . Intensive investigations in numerous
laboratories have demonstrated that these universally conserved
proteins participate in a wide variety of protein metabolic events in
both normal and stressed cells, including protein
folding(8, 9) , protein trafficking across
intracellular membranes(10, 11) , proteolysis, protein
assembly, as well as disassembly of protein aggregates and multiprotein
structures (reviewed in (12) and (13) ). Because
several of these DnaJ and DnaK family members have the capacity to
modulate polypeptide folding and unfolding, they have been classified
as molecular chaperones(14) .
The available evidence
indicates that DnaJ, DnaK, and GrpE of E. coli often cooperate
as a chaperone team to carry out their physiological roles. Each of
these three proteins functions in (i) regulation of the bacterial heat
shock response(15, 16) ; (ii) general intracellular
proteolysis(17) ; (iii) folding of nascent polypeptide chains,
maintaining proteins destined for secretion in a
translocation-competent state, and disassembly and refolding of
aggregated proteins (reviewed in (18) ); (iv) flagellum
synthesis (19) ; (v) replication of coliphages and P1 and
replication of the F episome(20) . DnaK, the primary Hsp70
homologue of E. coli(21) , is believed to play a
central role in these processes. Like other members of the Hsp70
family, DnaK possesses a weak ATPase activity (22) . The DnaK
ATPase activity is stimulated by the DnaJ and GrpE heat shock
proteins(23, 24) , as well as by many small peptides
that are at least 6 amino acids in length(24, 25) .
Peptide interactions with DnaK may be representative of the binding of
Hsp70 proteins to unfolded or partially folded polypeptides.
In
contrast to the situation for DnaK and Hsp70 proteins, relatively few
investigations have focused on DnaJ or other members of the Hsp40
family. It is known from in vitro studies of DNA
replication that DnaJ participates along with DnaK in the assembly and
disassembly of nucleoprotein structures that form at the viral
replication
origin(26, 27, 28, 29, 30) .
DnaJ may play a dual role in this and other Hsp-mediated processes. In
addition to activating ATP hydrolysis by DnaK, DnaJ, by binding first
to multiprotein assemblies or nascent polypeptides, may also assist
DnaK by facilitating its interaction with polypeptide
substrates(26, 30, 31, 32, 33, 34) .
Comparisons of the amino acid sequences of DnaJ family members has led to the identification of three conserved sequence domains in E. coli DnaJ(35) . These sequence domains, proceeding from the amino terminus, are: 1) a highly conserved 70-amino acid region, termed the J-region, that is found in all DnaJ homologues; 2) a 30-amino acid sequence that is unusually rich in glycine and phenylalanine residues; and 3) a cysteine-rich region that contains four copies of the sequence Cys-X-X-Cys-X-Gly-X-Gly, where X generally represents a charged or polar amino acid residue. The COOH-terminal portion of E. coli DnaJ, comprising residues 210-376, is not well conserved.
To investigate the functional roles of the conserved sequence domains of DnaJ, we constructed a series of recombinant plasmids that express truncated DnaJ proteins, each of which carries a deletion of one or more of the conserved sequence elements. Following purification, each deletion mutant protein was examined for its capacity to activate ATP hydrolysis by DnaK. Our results indicate that the J-region and the Gly/Phe-rich segment of DnaJ must both be present in cis in the DnaJ deletion mutant protein to achieve stimulation of DnaK-mediated ATP hydrolysis. We have, however, discovered that the J-region alone is capable of activating ATP hydrolysis by DnaK if it is supplemented in trans with small peptides that have high affinity for the polypeptide-binding site on DnaK. We discuss the relevance of these findings for protein metabolic events mediated in part by cooperative action of Hsp70 homologues and DnaJ-like proteins.
Plasmids carrying the wild type dnaJ gene or a dnaJ deletion mutant gene were constructed by cloning a DNA fragment
produced by polymerase chain reaction (PCR)-mediated amplification of E. coli genomic DNA with the aid of synthetic oligonucleotide
primers. The sequences of the forward primers used were:
oligonucleotide A
(5`CCACCGGATCCAGGAGGTAAAAATTAATGGCTAAGCAAGATTATTAC-3`), oligonucleotide
B (5`-CCACCGGATCCAGGAGGTAAAAATTAATGGCTGCGTTTGAGCAAGGT-3`), and
oligonucleotide C
(5`CCACCGGATCCAGGAGGTAAAAATTAATGCGTGGTCGTCAACGTGCG-3`). Each forward
primer contained a BamHI recognition site, a consensus
ribosome binding site, and an ATG initiation codon juxtaposed to dnaJ coding sequence (underlined in the primer sequences
listed above). These coding sequences correspond to dnaJ nucleotides 1-21 for primer A; dnaJ nucleotides
217-234 for oligonucleotide B; and dnaJ nucleotides
318-333 for primer C. The sequences of the reverse primers were:
oligonucleotide D (5`-CCACCTCTAGACTGCAGGTCGACATCTTAGCGGGTCAGGTCGTC-3`),
oligonucleotide E (5`-CCACCTCTAGACTGCAGGTCGACATCTTACTCAAACGCAGCATG-3`),
and oligonucleotide F
(5`-CCACCTCTAGACTGCAGGTCGACATCTTAACGTCCGCCGCCAAA-3`). Each reverse
primer contains a PstI recognition site, the complement of two
tandem translation stop codons, and the complement of dnaJ coding sequence (underlined). The sequences complementary to dnaJ coding sequence were: oligonucleotide D, complement of dnaJ nucleotides 1131-1114; primer E, complement of dnaJ nucleotides 225-211; and oligonucleotide F,
complement of dnaJ nucleotides 318-304. PCR
amplification was performed in a reaction mixture (100 µl)
containing 120 ng of high molecular weight E. coli DNA, 100
pmol each of one forward and one reverse primer, 50 µM of
each of the four dNTPs, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl, 0.01% gelatin, and 2.5
units of Amplitaq DNA polymerase.
Plasmid pRLM232 was constructed by PCR amplification of the entire E. coli dnaJ coding sequence, using primers A and D. Following amplification, the PCR fragment was digested with BamHI and PstI, and ligated to pRLM76 DNA which had been similarly digested at the unique BamHI and PstI sites present in the polylinker carried by this vector. The DNA in the ligation mixture was transformed into E. coli strains RLM569 and PK102. Ampicillin-resistant clones were selected at 30 °C and screened for their capacity to overproduce a polypeptide of the size of full-length DnaJ protein (i.e. 41 kDa) when grown at 42 °C. Plasmid pRLM233 (a pRLM76 derivative that expresses DnaJ1-75) was constructed and identified as above, except that the primers A and E were used for the initial PCR amplification and that the ampicillin-resistant transformants were screened for their capacity to thermally induce overproduction of a protein of 9 kDa. Plasmids pRLM234, pRLM238, and pRLM239, i.e. pRLM76 derivatives for expression of DnaJ1-106, DnaJ73-376, and DnaJ106-376, respectively, were constructed and identified by similar procedures. Several plasmids of each type were selected for DNA sequence analysis.
Frozen cell suspensions (58 ml, equivalent to about 8 g of
cell paste) were thawed and cell lysis was induced by three cycles of
quick-freezing in liquid nitrogen and thawing in water at 4 °C. Egg
white lysozyme was added to a final concentration of 0.1 mg/ml to the
cell lysate, which was further incubated at 4 °C for an additional
30 min. The lysate was supplemented with 20 ml of lysis buffer and
particulate material was removed by centrifugation for 60 min at 40,000
rpm in a Beckman 45Ti rotor (120,000 g). All
subsequent purification steps for DnaJ and each of the DnaJ deletion
mutant proteins were carried out at 0-4 °C. To the
supernatant (Fraction I, 70 ml), ammonium sulfate was slowly added to
40% saturation (0.226 g of ammonium sulfate/ml of supernatant), and the
suspension was stirred for 30 min at 4 °C. The precipitate that
formed was removed by centrifugation at 30,000
g for 1
h. Solid ammonium sulfate was added to the supernatant to 55%
saturation (0.089 g of ammonium sulfate/ml of supernatant), and, after
stirring for 30 min, the precipitate was collected by centrifugation at
30,000
g for 1 h. The pelleted precipitate was
resuspended in 50 ml of buffer B and dialyzed for 16 h against 4 liters
of buffer B. The dialyzed protein (Fraction II, 130 mg, 60 ml) was
applied to a Bio-Rex 70 column (5
10 cm) that had been
equilibrated with buffer B. The column was washed with 600 ml of buffer
B and bound proteins were subsequently eluted with a 800-ml linear
gradient of 0.15-1.0 M NaCl in buffer B at a flow rate
of 3 column volumes per h. The peak DnaJ-containing fractions were
pooled (150 ml;
0.42 M NaCl) and concentrated to 40 ml
using an Amicon stirred cell concentrator fitted with a PM-10 membrane.
The concentrated protein sample (Fraction III, 50 mg, 40 ml) was
dialyzed for 16 h against 4 liters of buffer C and applied to a P-11
phosphocellulose column (2.4
11 cm), that had been equilibrated
with buffer C. The column was subsequently washed with 150 ml of buffer
C and bound proteins were eluted with a 250-ml linear gradient of
0.15-1.0 M NaCl in buffer C at a flow rate of 1.4 column
volumes per h. DnaJ eluted at approximately 0.35 M NaCl. The
primary DnaJ-containing fractions were pooled (80 ml) and concentrated
to 40 ml with an Amicon concentrator as described above. This sample
was dialyzed against 2 liters of buffer C to produce Fraction IV (35
mg, 40 ml). Fraction IV protein was applied to a hydroxyapatite column
(2.4
11 cm) equilibrated with buffer C. The column was washed
with 150 ml of buffer C and bound protein was eluted with a 250-ml
linear gradient of 120-500 mM potassium phosphate in
buffer C at a flow rate of 1.4 column volumes/h. DnaJ eluted at
approximately 0.4 M potassium phosphate. Fractions containing
the predominant portion of DnaJ protein were pooled (40 ml),
concentrated to 10 ml in an Amicon apparatus, and dialyzed extensively
against buffer A. The dialyzed sample (Fraction V, 20 mg, 10 ml) was
diluted with an equal volume of buffer E, containing 2 M ammonium sulfate, to produce a conductivity equivalent to that of
buffer D. This sample was applied to a Pharmacia-Biotech
Butyl-Sepharose 4B column (2.4
11 cm) equilibrated with buffer
D. DnaJ was eluted with a 200-ml linear gradient of 100% buffer D to
100% buffer E, followed by 50 ml of buffer E, at a flow rate of 1
column volume/h. DnaJ eluted at approximately 95-100% buffer E.
Fractions containing DnaJ at greater than 90% purity, as analyzed by
SDS-PAGE, were pooled (30 ml) and concentrated to 10 ml using an Amicon
apparatus (Fraction VI, 10 mg, 10 ml). This protocol routinely produces
approximately 1.2 mg of DnaJ at greater than 90% purity per gram of
cell paste.
The physical properties of DnaJ73-376 and
DnaJ106-376 are similar to those of wild type DnaJ. Consequently,
these DnaJ deletion mutant proteins could be purified by using a
slightly modified version of the purification protocol used for DnaJ.
Frozen cells were resuspended in lysis buffer and lysed as described
above, except that the lysis buffer also contained 0.1%
octyl--D-glucopyranoside and 1 mM phenylmethylsulfonyl fluoride and that the cell lysate was mixed
gently for 12 h on a shaker at 4 °C. The lysate was centrifuged at
120,000
g for 1 h and the supernatant was supplemented
with ammonium sulfate to 40% saturation (0.226 g/ml of supernatant).
The precipitated protein was collected by centrifugation at 30,000
g for 1 h. The protein pellet was dissolved in 50 ml
of buffer A and dialyzed extensively against buffer B. All of the
remaining purification steps were identical to those used for purifying
wild type DnaJ. The final preparations of DnaJ73-376 and
DnaJ106-376 deletion mutant proteins were estimated to be greater
than 90% pure. They were quick-frozen in liquid nitrogen and were
stored frozen at -80 °C.
Figure 1:
Electrophoretic analysis of purified
DnaJ and DnaJ deletion mutant proteins before and after treatment with
papain. Wild type DnaJ protein (J1-376) and various DnaJ deletion
mutant proteins, including DnaJ1-75 (J1-75),
DnaJ1-106 (J1-106), DnaJ73-376 (J73-376), and
DnaJ106-376 (J106-376) (10 µg each), were digested with
papain (1% w/w) for 10 min at 30 °C, where indicated. The
undigested proteins as well as the polypeptide fragments produced by
papain proteolysis were resolved by electrophoresis in a 10-20%
gradient SDS-polyacrylamide gel and visualized by staining with
Coomassie Brilliant Blue R-250. The arrows indicate the
migration positions of DnaJ (41 kDa) and the 30- and 9-kDa stable
papain-resistant proteolytic fragments. The molecular weight markers (M) applied to the first and last lanes were: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa),
carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and
-lactalbumin (14.4 kDa).
To obtain a more refined estimate
of the positions of the COOH termini in each papain-resistant fragment
of DnaJ, we digested DnaJ with papain again, purified each polypeptide
by reverse-phase chromatography, and subjected each fragment to MALDI.
This analysis revealed that the small NH-terminal J-region
fragment was actually a series of fragments ranging in mass from
approximately 8770 to about 9900 Da. Comparison to the known sequence
of DnaJ indicates that the smaller protease-resistant fragments consist
of polypeptides containing DnaJ amino acid residues 2-75 through 2-89.
More prolonged digestion of a related polypeptide (DnaJ2-106, see
below) with papain yielded polypeptides whose masses were approximately
equivalent to DnaJ2-75 and DnaJ2-78. Thus, extensive papain
treatment of DnaJ results in nearly complete digestion of the
Gly/Phe-rich segment (amino acids 77-107). We conclude that the
papain-resistant structural domain encoded by the J-region corresponds
to DnaJ2-75 (Fig. 2). The removal of the
NH
-terminal methionine of DnaJ, however, is not the result
of papain action, but rather seems to be due to post-translational
processing in vivo, since our sequence analysis indicated that
alanine 2 is the NH
-terminal amino acid of purified DnaJ.
Figure 2:
Schematic representation of the partial
proteolysis of DnaJ protein with papain. The linear map of DnaJ protein
is depicted at the top. The positions of the major conserved
sequence elements are indicated, including the J-region, the glycine-
and phenylalanine-rich segment (G/F), and the cysteine-rich motifs
(Cys-rich). Papain treatment of wild type DnaJ produces 9- and 30-kDa
stable proteolytic fragments that retain the sequence elements
depicted. The NH-terminal amino acid sequences, in the
standard one-letter code, of each papain-resistant DnaJ fragment are
shown. The sizes of the proteolytic fragments were determined by laser
desorption mass spectrometry (see ``Experimental Procedures''
and the text for details). N, amino terminus; C,
carboxyl terminus.
The larger COOH-terminal polypeptide produced by the repeat papain
digestion of DnaJ was found by MALDI analysis to have a mass centered
about 30,115 Da (data not shown). If it is assumed that the COOH
terminus of DnaJ is resistant to proteolytic cleavage by papain, then
this preparation of the COOH-terminal papain fragment seemingly
includes DnaJ residues 99-376 (M =
30,130). More prolonged digestion of DnaJ with papain results in a
COOH-terminal polypeptide of approximately 29 kDa that has DnaJ residue
112 at its amino terminus, as revealed by NH
-terminal
sequence analysis. We conclude that the initial papain cleavages of
DnaJ occur between amino acid residues 80 and 100 and that the
remainder of the Gly/Phe-rich segment of this heat shock protein is
excised following more extensive papain treatment (Fig. 2).
Figure 3: Linear maps of DnaJ protein and DnaJ deletion mutant proteins. The schematic maps indicate which conserved DnaJ sequence motifs are present in each polypeptide. The sequence motifs are explained in the text and in the legend to Fig. 2. The open rectangle on the right side of the maps of the DnaJ, DnaJ73-376, and DnaJ106-376 proteins represents amino acid residues 205-376 of DnaJ, a region that does not share substantial homology with sequences of other members of the DnaJ family.
DnaJ1-75 was designed for
investigations of the functional significance of the highly conserved
J-region. PCR was used to amplify the sequence for dnaJ codons
1-75. In this amplification, as well as in other amplifications
of segments of the dnaJ gene by PCR, one of the primers
included a ``hang-off'' sequence encoding a consensus E.
coli ribosome binding site and an ATG initiator codon. Similarly,
the second primer used in the PCR amplification included a hang-off
sequence encoding the complement of two tandem stop codons. These
``3`'' primers were designed such that tandem stop codons
were juxtaposed, in the proper reading frame, to the 3` terminus of dnaJ coding sequences in the amplified DNA. The PCR products
were inserted into the polylinker site on pRLM76, an expression vector
that provides thermoinducible expression of genes cloned downstream
from a p
promoter present on the plasmid.
The resulting plasmid, pRLM233, was transformed into strain PK102, a dnaJ deletion mutant of E. coli(41) .
Induction of expression of the cloned gene fragment by aeration at 42
°C of cells harboring pRLM233 resulted in production of
DnaJ1-75 (J1-75) to amounts greater than 10% of the total
cellular protein (data not shown).
The DnaJ1-106 (J1-106) deletion mutant protein was designed for investigations of the functional significance of the Gly/Phe-rich sequence distal to the J-region. As for J1-75, a pRLM76-derivative that expresses J1-106 was constructed (pRLM234). Overexpressed J1-106 protein, like J1-75, was highly soluble and constituted approximately 10% of the total cellular protein following induction. Both J1-75 and J1-106 were purified to greater than 95% homogeneity as described under ``Experimental Procedures'' (Fig. 1). Although J1-75 and J1-106 have the same relative electrophoretic mobility in a 10-20% gradient SDS-polyacrylamide gel (Fig. 1), these two polypeptides can be readily resolved by electrophoresis in a 12% acid-urea polyacrylamide gel (Fig. 4).
Figure 4: Resolution of DnaJ1-106 from DnaJ1-75 and papain-resistant fragments of DnaJ1-106 by acid-urea polyacrylamide gel electrophoresis. DnaJ1-75 (J1-75) and DnaJ1-106 (J1-106) proteins (10 µg each) were digested with papain at 30 °C, as described under ``Experimental Procedures,'' for the indicated times. The polypeptide products were electrophoresed through a 12% acid-urea polyacrylamide gel and visualized by staining with Coomassie Brilliant Blue R-250. The arrows show the migration positions of undigested DnaJ1-75 (8.9 kDa) and DnaJ1-106 (11.7 kDa).
Two additional DnaJ deletion mutant proteins,
J73-376 and J106-376, were designed for investigations of
the functional role the COOH-terminal end of DnaJ. Both polypeptides
contain the cysteine-rich motifs and the COOH-terminal end of DnaJ.
Although neither mutant protein contains the J-region, J73-376
does contain the Gly/Phe-rich segment that links the two
papain-resistant structural domains found in wild type DnaJ (Fig. 3). DNA sequences that encode J73-376 and
J106-376, as well as appropriate translation signals, were
inserted into the expression site on pRLM76 to produce plasmids pRLM238
(J73-376) and pRLM239 (J106-376). E. coli transformants harboring these plasmids were thermally induced and
the overexpressed J73-376 and J106-376 proteins were
purified to greater than 90% homogeneity as described under
``Experimental Procedures.'' We found that both of these
mutant proteins had to be purified from a dnaJ deletion mutant
(PK102), since purification of J73-376 and J106-376 from a dnaJ strain (RLM569) resulted in the
copurification of a protein that we deduce is wild type DnaJ protein,
based on its electrophoretic mobility in SDS-PAGE and its reactivity
with polyclonal antibodies elicited against purified DnaJ protein (data
not shown).
It is possible that one or more of the deletion mutant
proteins fails to fold into a stable tertiary structure. Therefore, we
probed the structural integrity of each DnaJ deletion mutant protein by
examining its sensitivity to partial proteolysis with papain.
J1-75 is apparently a compact, well-folded protein. It was not
noticeably affected by papain digestion (Fig. 4). In contrast,
the J1-106 polypeptide was converted by papain to a fragment that
comigrates with J1-75 during electrophoresis in a 12% acid-urea
polyacrylamide gel (Fig. 4). Mass spectrometry revealed that
papain-mediated proteolysis of the J1-106 deletion mutant protein
produced two primary fragments, one with a molecular mass of 8960 Da
and the other of mass 8713 Da. These probably correspond to J-region
fragments containing amino acid residues 2-78 (M = 8962) and 2-75 (M
=
8720), respectively.
We obtained evidence that the J73-376 and
J106-376 deletion mutant proteins had also folded properly.
Partial proteolysis of these mutant proteins with papain resulted in
the production of polypeptide fragments that comigrate with the 30-kDa
proteolytic fragment of wild type DnaJ during electrophoresis under
denaturing conditions (Fig. 1). Furthermore, both J73-376
and J106-376, like wild type DnaJ, bind Zn and
display extensive secondary structure as revealed by atomic absorption
and circular dichroism spectroscopy, respectively. (
)
Figure 5:
Effect
of DnaJ and DnaJ deletion mutant proteins DnaJ1-75 and
DnaJ1-106 on ATP hydrolysis by DnaK. The progress curves of ATP
hydrolysis by DnaK were defined under single turnover conditions (2.3
µM DnaK and 30 nM [-
P]ATP) at various concentrations of
DnaJ, DnaJ1-75 (J1-75), and DnaJ1-106 (J1-106)
as indicated. ATPase assays were performed as described under
``Experimental Procedures.'' These data were used to
determine the first-order single turnover rate constant for ATP
hydrolysis by DnaK at each concentration of DnaJ, DnaJ1-75, or
DnaJ1-106. The single turnover rate constant for DnaK alone was
determined to be approximately 0.04
min
.
It has been suggested (48) that the highly conserved J-region, which we have shown is
essentially equivalent to the NH-terminal structural domain
of DnaJ, interacts with DnaK. We used the single turnover ATPase assay
to determine if the J-region is both necessary and sufficient for
stimulation of DnaK's ATPase activity. Purified J1-75
failed, even at very high concentrations, to produce any detectable
activation of the DnaK ATPase activity (Fig. 5). We next
examined whether J1-106, which contains both the J-region and the
Gly/Phe-rich segment, had any capacity to stimulate ATP hydrolysis by
DnaK. In striking contrast to J1-75, J1-106 is capable of
stimulating DnaK's intrinsic ATPase activity (Fig. 5).
However, the interaction of DnaJ1-106 with DnaK is clearly
deficient in some respects. At saturation, J1-106 yielded a
significantly slower ATPase rate constant than did wild type DnaJ.
Moreover, the apparent K
for J1-106 was
determined to be approximately 4 µM (Table 1). This
concentration is about 20-fold higher than the concentration of wild
type DnaJ required for half-maximal stimulation of DnaK's ATPase
activity.
These results suggested to us that two conserved DnaJ
motifs, i.e. the J-region and the Gly/Phe-rich segment,
participate jointly in the activation of the ATPase activity of DnaK.
However, it was still possible that the Gly/Phe-rich segment alone is
responsible for DnaJ's capacity to activate the DnaK ATPase. It
is known in this regard that peptide C binds to DnaK in an extended
conformation (49) and that many short peptides of 7 to 9 amino
acids or longer are capable of stimulating the intrinsic ATPase
activity of DnaK as well as the intrinsic ATPase activities of its
eukaryotic counterparts in the Hsp70
family(24, 25, 46, 47) . For DnaK,
the level of stimulation depends on the peptide sequence and can be as
much as 30-fold (24) . ()To examine the potential
role of the Gly/Phe-rich segment in the DnaJ-mediated activation of the
DnaK ATPase, we synthesized a set of five overlapping peptides, each 15
amino acids in length, that span the entirety of the Gly/Phe-rich
segment and tested each for its capacity to serve as an effector of the
DnaK ATPase. Under single turnover conditions, all of these peptides
failed to produce a significant activation of the DnaK ATPase; no more
than a 2- or 3-fold stimulation of the ATPase rate was observed, even
at millimolar concentrations of peptide (data not shown). These results
lend additional support to the hypothesis that the J-region and the
Gly/Phe-rich segment collectively contribute to the DnaJ-mediated
activation of DnaK.
Two additional DnaJ deletion mutant proteins, J73-376 and J106-376 (Fig. 3), were studied to examine whether the cysteine-rich motifs and the COOH-terminal portion of DnaJ play any independent role in activation of DnaK's ATPase activity. Neither mutant protein contains the J-region, but J73-376 does contain the flexible Gly/Phe-rich segment in addition to the COOH-terminal structural domain of DnaJ. Our results indicate that neither J73-376 nor J106-376 (in the range between 0.1 and 20 µM) was capable of providing detectable stimulation of the intrinsic ATPase of DnaK ( Table 1and data not shown). The inability of J73-376 to activate the DnaK ATPase provides additional evidence that the Gly/Phe-rich segment and the J-region must be simultaneously present in the DnaJ polypeptide in order to achieve significant stimulation of ATP hydrolysis by the DnaK heat shock protein.
Figure 6:
DnaJ1-75 and peptide cooperate to
activate ATP hydrolysis by DnaK. ATPase assays were conducted under
single-turnover conditions as described under ``Experimental
Procedures'' and the legend to Fig. 5, except that each
reaction mixture also contained peptide C (500 µM) and
either DnaJ1-75 or DnaJ73-376 as indicated. The reaction
progress curves at each concentration of DnaJ1-75 or
DnaJ73-376 were used to determine the first-order single turnover
rate constants. The single turnover rate constant for ATP hydrolysis by
DnaK in the presence of 500 µM peptide C alone was
determined to be approximately 0.5 min. Neither
DnaJ1-75 nor DnaJ73-376 alone was capable of stimulating
ATP hydrolysis by DnaK under single turnover conditions (Table 1).
Figure 7:
Dependence of DNA replication in
vitro on DnaJ protein and DnaJ deletion mutant proteins
DnaJ1-75 and DnaJ1-106.
DNA replication assays were
performed as described under ``Experimental Procedures,''
except that the standard reaction mixture was supplemented with DnaJ or
with a DnaJ deletion mutant protein as indicated. The titration of wild
type DnaJ in this assay is incomplete due to the fact that the presence
of high levels (i.e. 1-2 µg) of wild type DnaJ
protein in the reaction mixture severely inhibited
DNA
replication (data not shown). Filled circles, DnaJ protein; filled squares, DnaJ1-106 protein; open
squares, DnaJ1-75 protein.
Figure 8:
Dependence of DNA replication in
vitro on DnaJ or DnaJ deletion mutant proteins DnaJ73-376 or
DnaJ106-376.
DNA replication assays were conducted as
described under ``Experimental Procedures,'' except that the
standard reaction mixture was supplemented with wild type DnaJ protein
or a DnaJ deletion mutant protein, as indicated. Filled
circles, DnaJ protein; open squares, DnaJ106-376
protein; filled squares, DnaJ73-376
protein.
Our investigation of the capacities of various DnaJ deletion
mutant proteins to stimulate ATP hydrolysis by the E. coli DnaK molecular chaperone has identified two regions of DnaJ that
mediate this activation. The first region consists of the highly
conserved J-region, located at the amino terminus of DnaJ. This
70-amino acid region, which is the signature sequence of each member of
the ubiquitous DnaJ (Hsp40) family of molecular chaperones, forms a
stable structural domain in DnaJ(50, 51) . The
Gly/Phe-rich region of DnaJ, a polypeptide segment that links the
NH- and COOH-terminal domains of DnaJ, also plays a central
role in the activation of ATP hydrolysis by DnaK. The hypersensitivity
of this segment to proteolysis, as well as the preponderance of glycine
residues in this region, suggest that this polypeptide linker segment
is both relatively unstructured and highly flexible. This conclusion is
consistent with the results of a recent NMR structure determination of
an amino-terminal DnaJ fragment (DnaJ2-108) which found that the
Gly/Phe-rich region was flexibly disordered in solution(50) .
Mutant DnaJ proteins that contain either the J-region or the linker
region alone are incapable of stimulating ATP hydrolysis by DnaK (Table 1). Furthermore, in preliminary experiments, we have not
observed any capacity of these two regions of DnaJ to complement one
another and stimulate DnaK under conditions where the required regions
are located in trans on separate polypeptides (e.g. when DnaJ1-75 and DnaJ73-376 (Fig. 3) are mixed
together with DnaK). A truncated DnaJ polypeptide produced detectable
activation of DnaK's ATPase activity only when both the J-region
and the Gly/Phe-rich linker region were present in cis on the
same DnaJ deletion mutant polypeptide, as with, for example,
DnaJ1-106.
We sought to localize more definitively the amino acid sequence or sequences present in the Gly/Phe-rich linker region of DnaJ that contribute to the activation of ATP hydrolysis by DnaK. However, none of a series of overlapping 15-amino acid synthetic peptides corresponding to subsections of this linker region were found to provide significant activation of DnaK, whether or not the J-region domain (i.e. DnaJ1-75) was also present in the incubation mixture. This result was interesting, especially in view of the fact that previous studies have established that random peptides with as few as 6-9 amino acid residues are capable of stimulating the intrinsic ATPase activity of DnaK(24, 46, 47) . We have not determined if the synthetic DnaJ linker peptides simply fail to bind stably to DnaK or if, on the other hand, they bind to DnaK but fail to provoke a necessary response, e.g. a conformational change in DnaK needed to potentiate ATP hydrolysis.
Further exploration of the factors that influence ATP hydrolysis by DnaK led us to the conclusion that DnaK must simultaneously undergo two separate interactions to acquire optimal activation. One such interaction is with the J-region of its partner chaperone, DnaJ. But, as discussed above, the presence of the J domain alone has no discernible impact on ATP hydrolysis by DnaK. Thus, our results are not in agreement with a previous study that concluded that the J-region of DnaJ is both necessary and sufficient to stimulate ATP hydrolysis by DnaK(52) . We have shown that a second stimulatory interaction is required, one that involves binding of a peptide or protein substrate to the polypeptide-binding site on DnaK. Proteins, such as wild type E. coli DnaJ or DnaJ deletion mutant DnaJ1-106, that carry both the J-region and the Gly/Phe-rich linker segment can individually furnish in cis both interactions needed for activation of the DnaK ATPase(52) . We have demonstrated, however, that the requisite stimulatory interactions with DnaK can also occur in trans. A combination of the J domain (DnaJ1-75) and any short peptide that has high affinity for the polypeptide binding site of DnaK produced an activation of the DnaK ATPase comparable to wild type DnaJ alone (e.g. compare the data in Fig. 5and Fig. 6). While peptide C alone can stimulate ATP hydrolysis by DnaK as much as 20-fold(24) , addition of the J-domain to a reaction mixture containing peptide C and DnaK resulted in a 15-20-fold further enhancement of the rate constant for ATP hydrolysis.
Considerable genetic and biochemical evidence has been accumulated in support of the idea that proteins of the DnaJ family functionally cooperate with specific Hsp70 proteins in all organisms to mediate protein folding, protein assembly, and disassembly events, and translocation of polypeptides across intracellular membranes. Although direct physical evidence for a stable protein-protein interaction between these two ubiquitous chaperone types has been observed only in a thermophilic bacterium thus far(53) , recently it was demonstrated that the primary E. coli Hsp70 protein, DnaK, does bind to DnaJ when ATP is present(54) .
Genetic suppression studies in the yeast Saccharomyces cerevisiae(55) , as well as subsequent biochemical and cell biological analysis(56, 57) , provide additional support for the occurrence of both functional and direct physical interactions in the endoplasmic reticulum (ER) between chaperone-like proteins of the Hsp70 and Hsp40 families, i.e. between Kar2p, a DnaK and BiP homologue, and Sec63p, a member of the DnaJ family, respectively. These two proteins are thought to play a central role in the translocation of polypeptides from the yeast cytosol into the ER(10, 11, 58, 59) . While the precise molecular role of ER-localized Hsp70 proteins in protein translocation remains to be defined, it is reasonable to assume that the translocation process takes advantage of the capacity of such molecular chaperones to couple ATP hydrolysis and binding to polypeptide binding and release. Although Sec63p is an integral membrane protein associated with the polypeptide translocation complex in the ER, it does contain a 70-amino acid J-domain that faces the ER lumen(60) . Interestingly, Sec63p does not contain a segment that is homologous to the Gly/Phe-rich linker polypeptide of DnaJ. Thus, it is highly probable that Sec63p itself, like the J-domain (DnaJ1-75), is capable of contributing only one of the two signals required for activation of ATP hydrolysis by the Kar2 Hsp70 protein. In reaching this conclusion, we make the presumption that the dual signal requirement for maximal ATP hydrolysis we identified for DnaK has been conserved during evolution in all primary Hsp70 family members.
If Sec63p indeed only contributes a J-domain to the
activation process for Kar2p, then what is the source of the second
signal? Since the missing signal involves interaction of a peptide or
polypeptide with the polypeptide-binding site on the COOH-terminal
domain of Kar2p (BiP), we suggest that it is the translocating
polypeptide itself that supplies the other required signal for
activation of the Kar2p ATPase. This proposal is consistent with the
polypeptide binding specificity of the Hsp70 COOH-terminal domain as
well as the presumed structure of translocating polypeptide chains. The
available evidence indicates that Hsp70 proteins prefer to bind to
extended polypeptide chains containing substantial hydrophobic
character(25, 47, 49, 61, 62, 63) .
Accordingly, translocating polypeptides associated with Sec63p and the
ER translocation apparatus would be expected to be in an unfolded or
partially folded state as they emerge from the lipid bilayer of the ER.
In our proposal, simultaneous interaction of a molecule of the
ATP-bound form of BiP (Kar2p) with both the translocating polypeptide
and Sec63p would activate ATP hydrolysis by BiP. Recent findings
suggest that such ATP hydrolysis by BiP would effectively lock the
polypeptide substrate onto a BiPADP enzyme
complex(34, 64) . Moreover, this stable
Hsp70-polypeptide interaction, mediated in part by the J-domain of
Sec63p, may render polypeptide translocation into the ER irreversible,
a role that has also been suggested for Hsp70-polypeptide interactions
that occur during protein translocation into
mitochondria(65, 66) . The translocating polypeptide,
presumably still in an unfolded or partially folded conformation, would
be anticipated to remain firmly bound to BiP until the ADP present on
the enzyme is exchanged for ATP(64) .
Since no GrpE
homologue in the ER lumen has yet been identified, this nucleotide
exchange step may be slow(23, 64) .
A number of
instances have been described where it is DnaJ, rather than DnaK, that
first binds to a protein substrate of this chaperone system. This
situation was initially found for binding of DnaJ to a nucleoprotein
preinitiation complex formed at the bacteriophage replication
origin (26, 27, 30) and for binding of DnaJ
to the P1 phage-encoded RepA replication initiator
protein(67) . DnaJ also has high affinity for the E. coli
heat shock transcription factor (31, 68) and there is experimental support for the
idea that DnaJ may bind to nascent polypeptides as an early step in
protein folding in
vivo(33, 69, 70) . In each of these
cases, it seems likely that DnaJ may play roles both in recruiting one
or more molecules of DnaK to the locale of the protein substrate and in
subsequently facilitating the action of DnaK on the substrate.
While
our data and that of others (52, 59, 71) provide clear biochemical
evidence that the J-domain is critical to the process of Hsp70
recruitment and activation, the potential involvement of the
Gly/Phe-rich segment of DnaJ in Hsp70 recruitment, suggested by the
findings in this report, draws attention to a possible mechanistic
problem. For example, we have concluded here that the Gly/Phe-rich
segment of DnaJ provides one of the signals for DnaK activation by
binding to the polypeptide binding site of DnaK. Thus, DnaK recruited
to close spatial proximity of a protein substrate via interactions with
DnaJ apparently would first have to release the Gly/Phe-rich segment of
DnaJ before it could bind to its protein substrate. Our biochemical
studies are consistent with this pathway for DnaK action. The inability
of any of the synthetic peptides derived from the DnaJ Gly/Phe-rich
region to stimulate ATP hydrolysis by DnaK to a significant extent
suggests that the interaction of the DnaJ Gly/Phe-rich region with DnaK
must be both weak and transient. Perhaps the interaction between DnaK
and the J-domain present in the DnaJ-substrate complex is sufficiently
strong to keep DnaK from dissociating completely from DnaJ until DnaK
has had the opportunity to bind to the protein substrate. Moreover, a
prediction of this model is that binding of DnaK to the protein
substrate would be expedited by the high effective concentration of the
substrate which would arise because both DnaK and the substrate are
tethered to the same molecule of DnaJ. If it is presumed that DnaK is
bound to the NH-terminal J-domain and that the protein
substrate interacts with the COOH-terminal domain of DnaJ, it is
possible that the conformational flexibility of the Gly/Phe-rich
segment linking these two structural domains of DnaJ is an important
factor contributing to the optimization of DnaK-substrate interactions.
Wall et al.(68) have recently suggested a similar
model, as well as other possible scenarios, to explain the properties
of a DnaJ deletion mutant protein that is missing the Gly/Phe-rich
linker region.
We have provided evidence that a fragment of DnaJ
consisting of the amino-terminal 105 amino acid residues is capable of
activating ATP hydrolysis by DnaK. Nevertheless, our results indicate
that the COOH-terminal domain of wild type DnaJ must play some role in
the activation process. For example, the maximal rate constant of ATP
hydrolysis by DnaK elicited by DnaJ1-106 saturates at a level
more than 5-fold lower than that produced by wild type DnaJ (Fig. 5). Furthermore, the K for
DnaJ1-106 in this process (4 µM) is approximately
20-fold higher than the K
for wild type DnaJ ( Table 1and Fig. 5). This suggests that DnaK interacts more
effectively with the full-length DnaJ polypeptide than with
DnaJ1-106. However, The beneficial effect of the COOH-terminal
domain of DnaJ on the interaction with DnaK may be indirect. It is
conceivable that the COOH-terminal domain of DnaJ simply serves to lock
or position the two required elements, i.e. the J-domain and
the Gly/Phe-rich region, in a configuration that is optimal for
interaction with DnaK. On the other hand, we have not rigorously
excluded the possibility that the COOH-terminal domain of DnaJ directly
enhances interactions with DnaK by providing other sequence elements
that bind to the polypeptide binding site on DnaK. Our findings
suggest, however, that if such elements exist, they must have
relatively low affinity for the DnaK polypeptide-binding site. This
conclusion is based on our finding that the DnaJ106-376 and
DnaJ73-376 deletion mutant proteins, which each contain an intact
COOH-terminal domain, fail to complement DnaJ1-75 for activation
of ATP hydrolysis by DnaK.
It is interesting that the
DnaJ1-106 mutant protein can support initiation of DNA
replication in vitro, albeit at a much reduced level (52) (Fig. 7). The apparent specific activity of
DnaJ1-106 in this process is approximately 1000-fold lower than
that of wild type DnaJ (Table 1). None of the other deletion
mutant proteins described here could support detectable levels of
DNA replication at the highest concentrations tested (Table 1).
Thus, the minimal sequence elements of DnaJ required for initiation of
DNA replication include both the J-domain and the Gly/Phe-rich
linker segment, which apparently must both be present in cis.
Since these are the same two DnaJ sequence elements required for
activation of ATP hydrolysis by DnaK, it is conceivable that
DnaJ1-106 aids
DNA replication simply by converting DnaK
into a more active ATPase. Activated DnaK, presumably composed of a
complex of DnaJ1-106 and DnaK, may have an improved capacity,
because of its heightened ATPase activity, to bind directly to
nucleoprotein preinitiation structures formed at ori
.
This nonspecific route would, in effect, by-pass the normal initiation
pathway whereby DnaK is apparently recruited to bind at specific sites, i.e. at those locations where wild type DnaJ is already bound
to the preinitiation complex assembled at the
replication
origin(26, 27, 30) . The greatly lowered
specific activity of DnaJ1-106 in initiation of
DNA
replication may well reflect the inability of this truncated DnaJ
mutant to bind specifically to preinitiation nucleoprotein structures;
as a consequence, DnaJ1-106, unlike wild type DnaJ, is not
capable of directing DnaK to act at precise sites on specific substrate
molecules. A recent characterization of the properties of a similarly
truncated DnaJ polypeptide lends additional support to this
interpretation(71) . An amino-terminal fragment of DnaJ, DnaJ12
(equivalent to DnaJ2-108), was found to be capable of activating
DnaK to bind to one of its physiological substrates, the
heat shock transcription factor. Furthermore, in contrast to the
behavior of wild type DnaJ, the DnaJ12 mutant protein was reported to
be capable of activating DnaK to bind the
polypeptide in the absence of any prior interaction of the DnaJ12
protein itself with this heat shock factor.
There are two
discrepancies between the findings reported here and previously
published data that merit further discussion. First, in contradiction
to this report, it was previously concluded that the DnaJ J-domain
alone was both necessary and sufficient to activate ATP hydrolysis by
DnaK(52) . This inference was based on the properties of a DnaJ
deletion mutant protein, DnaJ12, composed of the first 108 amino acids
of DnaJ. The properties of the DnaJ12 mutant protein appear to be
nearly identical to those of the DnaJ1-106 protein described
here. It is evident that the mutant DnaJ protein used to reach the
earlier conclusion in fact contained not only the J-domain, but also
the essential Gly/Phe-rich region as well. Second, Wall et al.(68) have recently described a DnaJ deletion mutant
protein, DnaJ77-107, that is missing 31 amino acids covering
the entire Gly/Phe-rich region. These authors demonstrated that this
mutant protein, nevertheless, is still capable of activating the ATPase
activity of DnaK. One possible explanation for the difference between
our findings is that, as alluded to earlier, DnaJ may contain multiple
sequence elements capable of interacting with the polypeptide-binding
site on DnaK. In addition to the element reported here in the
Gly/Phe-rich segment, other potential DnaK interaction sites could
reside in the COOH-terminal structural domain of DnaJ. A second
possibility is that the random six amino acid linker, HMGSHM, that
replaced the Gly/Phe-rich segment as a consequence of the construction
of the DnaJ
77-107 deletion mutant protein(52) , can
itself serve as a polypeptide binding element for DnaK. Perhaps almost
any unstructured and flexible polypeptide chain of sufficient length (i.e. greater than 5 amino acids(24) ) covalently
linked to the J-domain will support productive interactions between
DnaJ and DnaK.