©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Bipartite Signaling Mechanism Involved in DnaJ-mediated Activation of the Escherichia coli DnaK Protein (*)

(Received for publication, November 30, 1995)

A. Wali Karzai Roger McMacken (§)

From the Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2)-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(2)-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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

Reagents and Materials

Reagents and materials and their sources were: papain (Worthington Biochemical); Hepes (Research Organics); ATP, ADP, DEAE-Sepharose, butyl-Sepharose 4B, and Mono-Q HR FPLC columns (Pharmacia Biotech); restriction enzymes (New England BioLabs); Amplitaq DNA polymerase (Perkin-Elmer); polyethyleneimine-cellulose thin layer chromatography sheets (EM Industries); Bio-Rex 70 (100-200 mesh) and hydroxyapatite HTP (Bio-Rad); P-11 Cellulose Phosphate (Whatman Lab Sales); [alpha-P]ATP (3000 Ci/mmol) (Amersham); stirred cell concentrator and PM-30 and YM-3 membranes (Amicon). All other biochemicals were from Sigma.

Bacteriophage and E. coli Replication Proteins

Bacteriophage and E. coli replication proteins except DnaJ and DnaK were prepared as described elsewhere (36) . The purification schemes for DnaJ protein and DnaJ deletion mutant proteins are described in this article. The purification of DnaK protein has been described previously(24) . DnaK protein elutes in three distinct peaks following chromatography on a Mono-Q resin. All ATPase reactions described in this report were carried out with peak I material, i.e. DnaK protein in the earliest eluting peak.

Buffers and Media

Buffer A is 50 mM Hepes/NaOH, pH 7.6, 2 mM DTT, (^1)0.15 M NaCl, and 10% (v/v) glycerol; buffer B is 50 mM Hepes/NaOH, pH 7.6, 2 mM DTT, 0.15 M NaCl, 0.1% beta-D-octyl glucoside, and 10% (v/v) glycerol; buffer C is 50 mM potassium phosphate, pH 6.8, 2 mM DTT, 0.15 M KCl, and 10% (v/v) glycerol; buffer D is 50 mM Hepes/NaOH, pH 7.6, 2 mM DTT, 0.15 M NaCl, 1 M ammonium sulfate, and 10% (v/v) glycerol; buffer E is 25 mM Hepes/NaOH, pH 7.6, 2 mM DTT, 0.1 M NaCl, and 10% (v/v) glycerol; buffer F is 50 mM Hepes/NaOH, pH 7.6, 2 mM DTT, 25 mM NaCl, and 10% (v/v) glycerol. SDS-PAGE sample buffer is 0.188 M Tris-HCl, pH 6.8, 2.0 M beta-mercaptoethanol, 0.01 mM bromphenol blue, 30% (v/v) glycerol. Lysis buffer is 50 mM Hepes/NaOH, pH 7.6, 2 mM DTT, 1 M NaCl, and 2 mM MgCl(2). Denaturation buffer is 50 mM potassium phosphate, pH 6.8, 0.15 M KCl, 10% (v/v) glycerol, 1 mM DTT, and 7 M guanidinium hydrochloride. Terrific broth (TB) medium is prepared by mixing at room temperature 900 ml of sterile broth (12 g of Difco BactoTryptone, 24 g of Difco BactoYeast Extract, 4 ml of glycerol, 900 ml of H(2)O) with 100 ml of sterile phosphate buffer (0.17 M KH(2)PO(4) and 0.72 M K(2)HPO(4))(37) .

Determination of Protein Concentration

The protein concentrations of samples containing partially purified proteins were determined by the method of Bradford(38) , using bovine -globulin as a standard. The concentrations of purified DnaJ and DnaJ deletion mutant proteins were determined in denaturation buffer, using their individual molar extinction coefficients ((M)) as determined by the method of Gill and von Hippel(39) . The concentration of GrpE was determined by a modification of the method of Lowry et al.(40) using bovine serum albumin as a standard. The concentration of DnaK was determined using the calculated molar extinction coefficient of the native protein, 15,800 M cm(24) .

Strains and Plasmids

Two E. coli strains were used for the expression of DnaJ and DnaJ deletion mutant proteins; RLM569 (C600, recA, hsdR, tonA, lac, pro, leu, thr, dnaJ) and PK102 (DeltadnaJ15), which carries a deletion of the primary portion of the dnaJ coding sequence(41) . Plasmid pRLM76 was used as the expression vector for DnaJ and DnaJ deletion mutant proteins. Plasmid pRLM76 is a derivative of plasmid pHE6 (42) that is deleted for the DNA sequence that encodes the amino-terminal portion of the N gene. Plasmid pRLM76 contains a polycloning linker downstream from a phage p(L) promoter. Thermosensitive cI857 repressor protein, which represses transcription from p(L) at 30 °C, is constitutively expressed from a mutant cI gene present in pRLM76. Expression of genes cloned into the polycloning linker of pRLM76 can be greatly induced by shifting the growth temperature of cells harboring the plasmid to 42 °C. Incubation at this temperature results in a rapid inactivation of cI857 repressor protein and leads to an enormous increase in transcription from the strong p(L) promoter. Plasmid pRLM76 was constructed as follows: plasmid pHE6 DNA was digested to completion with HincII and the 375-bp fragment carrying both the p(L) promoter and a portion of the N gene was isolated. This fragment was further digested with HaeIII to produce two fragments of 148 and 227 bp. The 148-bp fragment carrying the p(L) promoter was isolated and ligated to a 3573-bp fragment isolated from pHE6 DNA that had been digested with SmaI and partially digested with HincII. This ligation mixture was transformed into RLM569 and ampicillin-resistant clones that carried a 3.7-kilobase plasmid were identified. A plasmid having the p(L) promoter in the desired orientation (i.e. directing transcription across the polylinker sequence) was identified and named pRLM76 (3721 bp).

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(2), 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.

DNA Sequencing

The dnaJ gene region in plasmid clones containing the dnaJ gene or dnaJ deletion mutants was sequenced on both strands using the dideoxynucleotide chain termination method with modified T7 DNA polymerase (Sequenase) as described by the manufacturer (U. S. Biochemical). Plasmids free of nucleotide substitutions were selected for further analysis.

Expression and Purification of DnaJ, DnaJ73-376, and DnaJ106-376

E. coli RLM569 cells carrying a pRLM76 derivative that expresses DnaJ or a DnaJ deletion mutant protein were grown aerobically in a Fernbach flask at 30 °C in 700 ml of Terrific Broth to an optical density of 3.0 at 600 nm. The cultures were induced by the addition, with rapid mixing, of 300 ml of Terrific Broth that had been prewarmed to 70 °C. The cultures were aerated at 42 °C for 2-3 h and subsequently the cells were collected by centrifugation. The cell pellets were resuspended in 25 ml of lysis buffer, quick-frozen in liquid nitrogen, and stored at -80 °C.

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 times 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 times 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 times 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 times 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 times 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 times 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 times 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-beta-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 times 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 times 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.

Expression and Purification of DnaJ1-75

RLM1340 (RLM569/pRLM233) cells were grown, thermally induced, harvested, and lysed as described above for wild type DnaJ. A cell lysate from 8 g of cells was centrifuged at 120,000 times g for 1 h. The supernatant (Fraction I, 70 ml) was supplemented with ammonium sulfate to 75% saturation (0.476 g of ammonium sulfate per ml of supernatant), stirred at 4 °C for 30 min, and centrifuged at 30,000 times g for 1 h. The supernatant was concentrated to 25 ml, using an Amicon stirred cell concentrator fitted with a YM3 membrane, and dialyzed against 2 liters of buffer F (Fraction II, 80 mg, 30 ml). Fraction II was applied to a Bio-Rex 70 column (3.4 times 11 cm) equilibrated in buffer F and the column was subsequently washed with 300 ml of buffer F. Bound proteins were eluted with an 400-ml linear gradient of 0.025-0.7 M NaCl in buffer F at a flow rate of 3 column volumes per hour. DnaJ1-75 eluted at approximately 0.12 M NaCl. The fractions containing the primary portion of DnaJ1-75 polypeptide were pooled (80 ml) and concentrated to 30 ml in an Amicon apparatus (Fraction III, 30 mg). Fraction III protein was applied to a Bio-Rad hydroxyapatite column (2.4 times 11 cm) that had been equilibrated with buffer C. The column was washed with 150 ml of buffer C and eluted with a 250-ml linear gradient of 0.05-0.5 M potassium phosphate at a flow rate of 1.4 column volumes per h. DnaJ1-75 eluted at approximately 0.09 M potassium phosphate. Fractions containing DnaJ1-75 at greater than 95% purity were pooled (25 ml) and concentrated to 15 ml in an Amicon apparatus. This sample (Fraction IV, 20 mg, 15 ml) was quick-frozen in liquid nitrogen and stored at -80 °C.

Expression and Purification of DnaJ1-106

RLM1341 (RLM569/pRLM234) cells were grown, thermally induced, harvested, and lysed as described for wild type DnaJ. A cell lysate from 8 g of cells was centrifuged at 120,000 times g for 1 h. The supernatant (Fraction I, 70 ml) was supplemented with ammonium sulfate to 55% saturation (0.236 g of ammonium sulfate/ml of supernatant), stirred for 30 min, and centrifuged at 30,000 times g for 1 h. The supernatant, which contained the vast majority of the J1-106 polypeptide, was brought to 70% saturation with ammonium sulfate (0.093 g of ammonium sulfate/ml of supernatant) and was stirred for 30 min. The protein precipitate was collected by centrifugation at 30,000 times g for 1 h. The pellet was resuspended in 50 ml of buffer F and dialyzed against 2 liters of buffer F (Fraction II, 75 mg, 60 ml). Fraction II protein was applied to a Bio-Rex 70 column (3.4 times 11 cm) equilibrated with buffer E. Subsequently, the column was washed with 300 ml of buffer E and bound proteins were eluted with a 400-ml linear gradient of 0.025-1.0 M NaCl in buffer E at a flow rate of 3 column volumes/h. DnaJ1-106 eluted at approximately 0.15 M NaCl. The fractions containing the highest concentration of DnaJ1-106 were pooled (90 ml) and concentrated to 40 ml in an Amicon stirred cell concentrator fitted with a YM-3 membrane (Fraction III, 35 mg, 40 ml). Fraction III was dialyzed against 4 liters of buffer C and applied to a hydroxyapatite column (2.4 times 11 cm) equilibrated with buffer C. The column subsequently was washed with 150 ml of buffer C and bound proteins were eluted with a 250-ml linear gradient of 50-500 mM potassium phosphate in buffer C at 1.4 column volumes/h. DnaJ1-106 eluted at approximately 0.12 M potassium phosphate. Fractions containing DnaJ1-106 at greater than 95% purity were pooled (40 ml) and concentrated to 10 ml in an Amicon apparatus (Fraction IV, 25 mg, 10 ml). The preparation of DnaJ1-106 was quick-frozen and stored at -80 °C.

Single Turnover ATPase Assay

ATPase reaction mixtures (30 µl) contained 25 mM Hepes/KOH, pH 7.6, 11 mM magnesium acetate, 0.3 M potassium glutamate, 30 nM ATP (0.05 µCi of [alpha-P]ATP), 2.3 µM DnaK, and DnaJ or DnaJ deletion mutant protein as specified. All molar protein concentrations given in this paper are based on the molecular weight of a monomeric form of the protein calculated from the known amino acid sequences: DnaK, 69,100; DnaJ, 41,105; DnaJ 1-75, 8,851; DnaJ1-106, 11,746; DnaJ73-376, 32,618; and DnaJ106-376, 29,659. Prior to the addition of ATP as the final component, all reaction mixtures were preincubated at 25 °C for 2 min. The reaction was initiated by the addition of ATP and incubated at 25 °C. At each time point 15-µl portions were removed to tubes containing 2 µl of 1 N HCl. This treatment lowered the pH to between 3 and 4 and quenched the ATPase reaction (control experiments indicated that little or no additional hydrolysis of ATP occurred subsequent to the addition of HCl). Portions (4 µl) from each quenched reaction mixture were applied to polyethyleneimine-cellulose thin layer chromatography plates that had been prespotted with 1 µl of a mixture containing ATP and ADP (each at 20 mM). The plates were developed in 1 M formic acid and 0.5 M LiCl. The migration positions of ATP and ADP were visualized by short wave UV irradiation, and the level of each in the reaction mixture was determined by scintillation counting. The kinetic data obtained from the single turnover ATPase reactions were fit to a first-order rate equation using the nonlinear regression program, ``Enzfitter'' (Biosoft, Cambridge, UK). K(A) values, for activation under single turnover conditions of the ATP hydrolysis step in the DnaK ATPase reaction cycle by DnaJ and DnaJ deletion mutant proteins, were obtained from a replot of k values versus concentration of DnaJ or DnaJ deletion mutant protein. For each activator protein, at least five different activator concentrations (over a 100-fold range of concentration) were examined to generate the kinetic data used for the determination of the individual K(A) values.

DNA Replication Assay

The in vitro assays for DNA replication were performed essentially as described(36) . The reaction mixture (30 µl) contained 40 mM Hepes/KOH, pH 7.6, 0.2 M potassium glutamate, 11 mM magnesium acetate, 5 mM ATP, 180 µM each of dATP, dCTP, and dGTP, 80 µM [^3H]dTTP (at 400-800 cpm/pmol of dTTP), 215 ng of supercoiled ori plasmid DNA (pRLM4), 195 ng of O protein, 100 ng of P protein, 175 ng of DnaB helicase, 540 ng of single-stranded DNA binding protein, 100 ng of primase, 80 ng of DNA polymerase III holoenzyme, 230 ng of GyrA protein, 240 ng of GyrB protein, and either 5.4 µg of DnaK protein (in the absence of GrpE protein) or 1.4 µg of DnaK protein and 20 ng of GrpE protein. Wild type DnaJ protein or DnaJ deletion mutant proteins were added to the replication assay as indicated. Following assembly of the replication reaction mixture, it was incubated for 40 min at 30 °C. The amount of DNA synthesis was determined by measuring the level of [^3H]dTMP that had been incorporated into acid insoluble material, which was collected on a glass fiber filter (Whatman AH) and counted in a liquid scintillation counter.

Papain Digestion

Papain (50 µg/ml) was activated by incubation for 15 min at 37 °C in a buffer containing 50 mM MES, pH 6.5, 1 mM DTT, 5 mM cysteine-HCl, and 0.1 mM beta-mercaptoethanol. DnaJ protein and DnaJ deletion mutant proteins were treated with activated papain, at 1% (w/w) papain:DnaJ protein, at 30 °C for varying times as indicated. Proteolytic digestion was stopped by the addition of an excess of E64, a papain inhibitor(43) . Samples (30 µl) were mixed with 70 µl of SDS-PAGE sample buffer, boiled, and analyzed by electrophoresis in a SDS-polyacrylamide gel as described below. Undigested proteins and proteolytic polypeptide fragments were visualized by staining the gel with Coomassie Brilliant Blue R-250.

Gel Electrophoresis and Amino Acid Sequence Analysis

Protein samples were mixed with an equal volume of SDS-PAGE sample buffer, boiled for 5 min, and subjected to electrophoresis in a 10-20% gradient SDS-polyacrylamide gel as described by Laemmli(44) . Acid/Triton X-100/urea gel electrophoresis (45) was performed in 12% polyacrylamide gels. For determination of amino acid sequences, proteins were blotted from SDS-polyacrylamide gels onto Immobilon-P transfer membrane filters (polyvinylidene difluroide membrane filters, Millipore). The protein bands were visualized by staining the filter with Coomassie Brilliant Blue R-250. The protein bands of interest were excised and each polypeptide was subjected, while still bound to the filter, to NH(2)-terminal amino acid sequence analysis using a modified Edman protocol. Amino acid sequence analysis was performed by the Johns Hopkins University Peptide Synthesis Facility.

Peptide Synthesis

Peptide C, peptide NR, and peptides corresponding to portions of the Gly/Phe-rich segment of DnaJ were synthesized by solid phase peptide synthesis at the Johns Hopkins University Peptide Synthesis Facility. The amino acid sequence of peptides C and peptide NR are: peptide C, KLIGVLSSLFRPK(46) ; and peptide NR, NRLLLTG(47) . The amino acid sequences of the peptides from the Gly/Phe-rich region of DnaJ are: peptide 1 (amino acids 68-82), QYGHAAFEQGGMGGG; peptide 2 (amino acids 78-92), GMGGGGFGGGADFSD; peptide 3 (amino acids 88-102), ADFSDIFGDVFGDIF; peptide 4 (amino acids 98-112), FGDIFGGGRGRQRAA; peptide 5 (amino acids 108-122), RQRAARGADLRYNMQ. Freshly lyophilized peptide preparations were used to prepare stocks of defined concentrations.

Matrix-assisted Laser Desorption/Ionization Mass Spectral Analysis (MALDI-MS) of Proteins and Polypeptides

MALDI-MS analysis of DnaJ was performed by the Middle Atlantic Mass Spectroscopy Facility (Johns Hopkins University School of Medicine) with a Kratos Kampact III linear time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm). Protein samples (20 µg), consisting of DnaJ or papain-resistant fragments of DnaJ, were prepared for mass spectral analysis using Bond-Elute disposable, solid phase extraction, C8 columns, according to the manufacturer's specifications (Varian Inc.). Briefly, 100 µl (20 µg) of protein sample was loaded onto a 0.5-ml Bond Elute C8 column equilibrated in buffer I (0.1% (v/v) trifluoroacetic acid in deionized H(2)O). The column was washed with 2.0 ml of buffer I and the protein sample was eluted with 500 ml of buffer I containing 95% aqueous acetonitrile. The eluted protein sample was dried in a Speed-Vac centrifugal concentrator and redissolved in 20 µl of buffer I containing 20% aqueous acetonitrile. The sample, or analyte (0.3 µl), was deposited on a sample site of a 20-site stainless steel slide that contained 0.3 µl of a saturated solution of the matrix (3,5-dimethoxy-4-hydroxycinnamic acid; 207.8 Da) in 50:50 (v/v) ethanol:water. The analyte-matrix solution was air-dried and the slide was subsequently inserted into the mass spectrometer. The spectra acquired represent the accumulation of data collected from 45 laser shots.


RESULTS

Partial Proteolysis of DnaJ

Analysis of the amino acid sequences of the DnaJ heat shock protein family indicates that there are two large regions that are conserved in multiple members of this family. The most highly conserved region is the 70 amino acid ``J-region,'' which is found in all members of the DnaJ family. The second region, which is present in several, but not all, DnaJ homologues, contains multiple Cys-rich motifs. We wished to determine if these conserved regions represent stable structural domains of DnaJ. A nonspecific protease, such as papain, can be useful for delimiting structural domains in proteins, since its enzymatic activity is generally ineffectual on stable secondary and tertiary structures in substrate proteins. DnaJ was digested with papain and the resulting polypeptide products were subjected to analysis by SDS-PAGE. Proteolysis of DnaJ with papain produced two stable fragments of approximately M(r) = 9,000 and 30,000 (Fig. 1, J1-376). Edman analysis of the NH(2)-terminal amino acid sequences of these fragments yielded the amino acid sequences AKQDYY for the 9-kDa fragment and GGRGRQ for the 30-kDa fragment, which correspond, respectively, to amino acids 2-7 and 104-109 of DnaJ. This demonstrates that the 9-kDa polypeptide encompasses the highly conserved ``J-region,'' whereas the 30-kDa fragment includes the cysteine-rich motifs of full-length DnaJ, but does not contain most of the Gly/Phe-rich segment of the native molecular chaperone.


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 alpha-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(2)-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(2)-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(2)-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(2)-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(r) = 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(2)-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).

Expression and Purification of DnaJ Deletion Mutant Proteins

Based on the identification of stable protease-resistant domains in DnaJ and on the location of sequence motifs that are conserved in multiple DnaJ homologues, we designed a series of DnaJ deletion mutant proteins to be used in structure-function studies. These mutant proteins, depicted schematically in Fig. 3, consist of the NH(2)-terminal J-region (DnaJ1-75) and the COOH-terminal, 30-kDa papain-resistant domain (DnaJ106-376) as well as derivatives of each that also contain the Gly/Phe-rich segment (DnaJ1-106 and DnaJ73-376, respectively).


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(L) 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(r) = 8962) and 2-75 (M(r) = 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. (^2)

Capacity of DnaJ Deletion Mutant Proteins to Stimulate the ATPase Activity of DnaK

We wished to determine if any of the DnaJ deletion mutant proteins retained any of the functional activities characteristic of wild type DnaJ, for example, its capacity to stimulate the weak intrinsic ATPase activity of DnaK(23, 24) . Because DnaJ stimulates the DnaK ATPase specifically at the hydrolytic step in the ATPase reaction cycle, (^3)the DnaK ATPase activity is especially sensitive to the presence of DnaJ when the ATPase assay is performed using single turnover conditions (i.e. when the concentration of DnaK greatly exceeds that of ATP). Under these conditions, DnaJ strongly stimulates ATP hydrolysis by DnaK (Fig. 5). The rate constant for ATP hydrolysis by DnaK is increased at least 200-fold at saturating levels of DnaJ, from 0.04 min to more than 8.5 min. These data yielded an apparent K(A) of 0.2-0.3 µM DnaJ for activation of the DnaK ATPase ( Fig. 5and Table 1).


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 [alpha-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(2)-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(A) 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) . (^4)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.

Activation of the DnaK ATPase by a Combination of DnaJ1-75 and Peptide

Polypeptides, such as full-length DnaJ and J1-106, that have a covalent linkage between the J-region and the Gly/Phe-rich segment, have the capacity to stimulate ATP hydrolysis by DnaK. The apparently unstructured nature of the Gly/Phe-rich segment, as judged by its sensitivity to proteolysis by papain, suggested the possibility that it interacts with the peptide-binding site on DnaK during DnaJ-mediated activation of the DnaK ATPase activity. We therefore sought to determine if a free peptide could replace the Gly/Phe-rich segment and complement the J-region for activation of DnaK. Incubation of DnaK with both J1-75 and any of the five synthetic peptides derived from the Gly/Phe-rich region produced no detectable stimulation of DnaK's ATPase activity under single turnover conditions (data not shown). However, when we used peptides, such as peptide C (24) or peptide NR(47) , that are capable of stimulating DnaK's ATPase activity, a significant further increase in the rate constant for ATP hydrolysis was observed in the presence of both J1-75 and peptide (Fig. 6). At saturating levels of J1-75 and peptide, the rate of ATP hydrolysis by DnaK was roughly equivalent to the enhanced DnaK ATPase rate elicited by the presence of wild type DnaJ. The maximal rate constant obtained in the presence of peptide and J1-75 was more than 200-fold greater than that for DnaK alone under similar conditions and more than 15-fold higher than that obtained when DnaK was supplemented with just peptide C or peptide NR. The concentration of J1-75 which produced half-maximal stimulation of DnaK's ATPase activity (i.e. apparent K(A)) in the presence of added peptide was determined to be 1.3 µM (Table 1). Under similar conditions, those DnaJ deletion mutant proteins that lack the 70-amino acid J-region, J73-376 and J106-376, were unable to activate hydrolysis of ATP by DnaK beyond that yielded by peptide alone ( Fig. 6and data not shown). Based on these data, we conclude that the DnaJ-mediated stimulation of ATP hydrolysis by DnaK occurs as the result of two separate interactions between these two molecular chaperones. ATP hydrolysis by DnaK apparently is maximally activated when it simultaneously interacts both with the amino-terminal domain of DnaJ and with a flexible peptide that can adopt an extended conformation. Wild type DnaJ protein provides both signals in the form of the J-region and the Gly/Phe-rich sequence, respectively.


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).



Replicative Potential of DnaJ Deletion Mutant Proteins

We have previously demonstrated that the DnaJ and DnaK molecular chaperones are absolutely required for the initiation of phage DNA replication in a system that is reconstituted with 10 highly purified and E. coli proteins(26, 36) . We examined various DnaJ deletion mutant proteins for their capacity to support DNA replication in the reconstituted multiprotein system. We wished to determine if there was a direct correlation between the capacity of a particular mutant protein to stimulate ATP hydrolysis by DnaK and its ability to support the initiation of bacteriophage DNA replication. Only a few nanograms of wild type DnaJ is sufficient to support maximal DNA replication in vitro (Fig. 7). In contrast, the DnaJ1-75 deletion mutant protein was inactive in this replication assay, even at very high protein concentrations (Fig. 7). DnaJ1-106, which contains both the J-region and the Gly/Phe-rich segment, supported limited DNA synthesis in the replication assay (Fig. 7). This response was extremely weak, however, requiring on a molar basis 1000-fold more J1-106 than full-length DnaJ to attain a similar level of replication. In related studies, we found that both J73-376 and J106-376 were inactive in the replication assay (Fig. 8). These data indicate that linkage of the J-region to the Gly/Phe-rich segment produces the minimal combination of DnaJ sequence elements that is capable of both activating ATP hydrolysis by DnaK as well as supporting initiation of DNA replication in vitro.


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.




DISCUSSION

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(2)- 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 BiPbulletADP 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) .^4 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(2)-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(A) for DnaJ1-106 in this process (4 µM) is approximately 20-fold higher than the K(A) 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, DnaJDelta77-107, 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 DnaJDelta77-107 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.


FOOTNOTES

*
This research was supported by National Institutes of Health Research Grants GM36526 and GM32253. We also acknowledge National Institutes of Environmental Health Sciences Center Grant ES-03819 for its support of the oligonucleotide synthesis facility. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 410-955-3949; Fax: 410-955-2926; rmcmacke{at}phnet.sph.jhu.edu.

(^1)
The abbreviations used are: DTT, dithiothreitol; bp, base pair(s); J1-75, DnaJ1-75; J1-106, DnaJ1-106; J73-376, DnaJ73-376; J106-376, DnaJ106-376; MES, 2-(N-morpholino)ethanesulfonic acid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption/ionization; ER, endoplasmic reticulum.

(^2)
A. W. Karzai and R. McMacken, manuscript in preparation.

(^3)
R. Russell, A. Mehl, R. Jordan, A. W. Karzai, and R. McMacken, manuscript in preparation.

(^4)
R. Jordan, R. Russell, A. Mehl, and R. McMacken, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Dr. John D. Roberts in the construction of plasmid pRLM76 as well as the help of Dr. Brian Learn in the preparation of figures. We thank Dr. Robert J. Cotter for his generosity in permitting us access to his time-of-flight mass spectrometer facility. We also thank Rick Russell, Dr. Andrew Mehl, and other laboratory members for discussions and for critical reading of this manuscript.


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