©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Conserved G/F Motif of the DnaJ Chaperone Is Necessary for the Activation of the Substrate Binding Properties of the DnaK Chaperone (*)

(Received for publication, July 19, 1994; and in revised form, November 9, 1994)

Daniel Wall (1) (2)(§) Maciej Zylicz (1) (3) Costa Georgopoulos (1) (2)

From the  (1)Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City, Utah 84132, the (2)Département de Biochimie Médicale, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Genève 4, Switzerland, and the (3)Division of Biophysics, Department of Molecular Biology, University of Gdansk, Kladki 24, Gdansk 80-822, Poland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The universally conserved DnaK and DnaJ molecular chaperone proteins bind in a coordinate manner to protein substrates to prevent aggregation, to disaggregate proteins, or to regulate proper protein function. To further examine their synergistic mechanism of action, we constructed and characterized two DnaJ deletion proteins. One has an 11-amino-acid internal deletion that spans amino acid residues 77-87 (DnaJDelta77-87) and the other amino acids 77-107 (DnaJDelta77-107). The DnaJDelta77- 87 mutant protein, was normal in all respects analyzed. The DnaJDelta77-107 mutant protein has its entire G/F (Gly/Phe) motif deleted. This motif is found in most, but not all DnaJ family members. In vivo, DnaJDelta77-107 supported bacteriophage growth, albeit at reduced levels, demonstrating that at least some protein function was retained. However, DnaJDelta77-107 did not exhibit other wild type properties, such as proper down-regulation of the heat-shock response, and had an overall poisoning effect of cell growth. The purified DnaJDelta77-107 protein was shown to physically interact and stimulate DnaK's ATPase activity at wild type levels, unlike the previously characterized DnaJ259 point mutant (DnaJH33Q). Moreover, both DnaJDelta77-107 and DnaJ259 bound to substrate proteins, such as , at similar affinities as DnaJ. However, DnaJDelta77-107 was found to be largely defective in activating the ATP-dependent substrate binding mode of DnaK. In vivo, the ability of the mutant DnaJ proteins to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind , in an ATP-dependent manner, and not with their ability to bind . We conclude, that although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved in an important manner in modulating DnaK's substrate binding activity.


INTRODUCTION

The DnaK (Hsp70 eukaryotic homolog), DnaJ (Hsp40 eukaryotic homolog), and GrpE molecular chaperones participate in a variety of biological process. Along with their central roles in protein folding and transport (Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993), they can modulate other aspects of protein function. For example, in bacteriophage -DNA replication these chaperones catalyze the disassembly of the DnaB helicase from the P inhibitory protein in a timely manner to allow DNA replication to initiate (Alfano and McMacken, 1989; Zylicz et al., 1989). In addition, chaperones may also regulate intramolecular protein interactions. For instance, presumably through a conformationally induced change, DnaK can ``activate'' the DNA-binding properties of the tumor suppressor p53 protein (Hupp et al., 1992).

Central to the mechanism of action of this chaperone machine, DnaJ stimulates the hydrolysis of DnaK-bound ATP (Liberek et al., 1991a). This stimulation correlates with an increased affinity of DnaK for certain substrates, i.e. (Liberek and Georgopoulos, 1993), RepA (Wickner et al., 1991), luciferase (Schröder et al., 1993), rhodanese (Langer et al., 1992), and P (Osipiuk et al., 1993; Zylicz, 1993). On the contrary, GrpE functions primarily as a nucleotide exchange factor for DnaK (Liberek et al., 1991a). In some instances, this function of GrpE has been shown to correlate with substrate release from DnaK, i.e. rhodanese (Langer et al., 1992) and P (Hoffmann et al., 1992; Osipiuk et al., 1993). Jointly, DnaJ and GrpE stimulate DnaK's ATPase activity 50-fold and likely facilitate the recycling of these chaperone proteins (Liberek et al., 1991a).

Previously we have established that the DnaJ12-truncated protein, containing only the amino-terminal 108 amino acids of DnaJ is necessary and sufficient to (i) stimulate DnaK's ATPase activity, (ii) regulate DnaK's conformational state in the presence of ATP, (iii) activate DnaK to bind in the presence of ATP and, (iv) elicit proper, albeit limited, chaperone function from DnaK for -DNA replication (Wall et al., 1994). (^1)These conclusions were based primarily on the findings that DnaJ12 possessed all these functions, while DnaJ259, which carries a single His Gln substitution at residue 33, lacked all of these activities. Here, we address the function of the G/F motif of DnaJ, which comprises amino acid residues 77-107.


MATERIALS AND METHODS

Bacteria and Plasmids

The dnaJ G/F internal deletion mutants were constructed, via polymerase chain reaction, using the primers pJ7 (5`-CCGGATCCCATATGTTGCTCAAACGCAGC-3`) and pJ8 (5`-CCG GATCCCATATGCGTCAACGTGCGGCGCG-3`) for DnaJDelta77-107 and pJ7 and pJ9 (5`-CCGGATCCCATATGGCAGACTTCAGCGATATT-3`) for DnaJDelta77-87. A flanking primer to the 5` of the dnaJ gene was used with pJ7, and a primer 3` of pJ8 and pJ9 was used to amplify the DNA. Primers pJ7, pJ8, and pJ9 all have BamHI and NdeI restriction sites engineered into their 5` tails (underlined). These polymerase chain reaction products were digested with BamHI, and the appropriate DNA fragments produced were ligated together, creating the desired deletions (and simultaneously substituting in the amino acids HMGSHM in the resulting gene products). The gel-purified products were then digested with Bpu1102 and SacI, and ligated into the corresponding sites of the pDW19dnaJ plasmid (Wall et al., 1994). The accuracy of the constructs was verified by DNA sequence analysis. To test for genetic complementation, the various mutant plasmid constructs were transformed into the host strains B178 DeltadnaJ::Kan^R (Wall et al., 1994) or MC4100 DeltadnaJ::Kan^R Ø(pgroE-lacZ) (Missiakas et al., 1993), by selecting for amp^R. Standard genetic (Miller, 1992) and molecular biology techniques (Sambrook et al., 1989) were followed as described or as recommended by the manufacturers.

Protein Purifications

The , DnaK, DnaJ, DnaJ259, and GrpE proteins were purified as described previously (Liberek et al., 1992; Wall et al., 1994; Zylicz et al., 1985, 1987, 1989). The GrpE and DnaJ proteins were generous gifts of Drs. A. Wawrzynow and B. Banecki, respectively. DnaJDelta77-107 was purified essentially as wild type DnaJ, except that the urea extraction step was replaced by increasing the ionic strength of the lysis buffer (1 M NaCl). The purity of all proteins was >90%. Protein concentrations were estimated by the Bio-Rad assay and sodium dodecyl sulfate-polyacrylamide electrophoresis, followed by Coomassie Blue staining.

Enzyme-linked Immunosorbent Assays (ELISA)^2

This is a modified procedure of that previously described (Marszalek and Kaguni, 1994). The indicated proteins were added to 96-well microtiter plates in phosphate-buffered saline (PBS) (Sambrook et al., 1989), in a 50-µl volume. After 1 h of incubation at room temperature, the solution was removed and the wells were washed three times with PBS containing 0.2% bovine serum albumin (BSA, 100 µl). The last wash solution was incubated for 1 h before removal. The wells were then washed with buffer A (25 mM HEPES, pH 7.6, 150 mM KCl, 25 mM NaCl, 5 mM MgCl(2), 0.1 mM EDTA, 1 mM dithiothreitol (and 0.1% Triton X-100), and the indicated protein(s) were added in 50 µl of buffer A for 30 min. Glutaraldehyde was added to 0.1% followed by a 10-min incubation at RT. The wells were then washed with buffer A followed by two washes of PBS, 0.2% BSA (100 µl). The indicated antibodies were then added in PBS, 0.2% BSA (100 µl) and incubated for 2 h at RT. Unbound protein was removed followed by three washes with PBS, 0.2% BSA. The secondary antibody was added in PBS, 0.2% BSA (100 µl) for 45 min. Following protein removal and three washes as described previously, colorimetric detection was done with the Bio-Rad TMB peroxidase enzyme immunoassay substrate kit. The reaction was stopped with an equal volume (100 µl) of 4 N sulfuric acid. Absorbance (490 nm) was measured with a Molecular Devices ThermoMax microplate reader.


RESULTS AND DISCUSSION

Sequence analysis of over 30 DnaJ family members from prokaryotic and eukaryotic organisms suggests that the amino-terminal 108 amino acids of DnaJ consists of two well-conserved domains or modules. The first 76 amino acids constitute the most highly conserved region of DnaJ that has been used as the ``signature'' sequence to identify additional DnaJ family members, and is thus called the J-domain (Bork et al., 1992; Silver and Way, 1993; Wall et al., 1994). The next 31 amino acids are rich in the amino acids Gly and Phe and are therefore referred to as the G/F motif or module (Fig. 1). Since this region is rich in gly residues, it has been suggested that the G/F motif functions as a linker between the J-domains and the rest of the DnaJ protein (Silver and Way, 1993). However, an amino acid alignment of this motif reveals a considerable amount of sequence conservation (Fig. 1). In particular, there is a nearly universally conserved Asp-Ile-Phe (DIF) motif (in two cases Ile has been replaced by Val, a conservative substitution). Escherichia coli's DnaJ has two additional DIF repeats (Fig. 1). The existence of these highly conserved residues suggests that this DIF motif may be important for the structure/function of DnaJ. The alignment also reveals a certain amount of flexibility in the number of Gly residues. Interestingly, some DnaJ family members do not contain this motif, although most do (Bork et al., 1992), suggesting that the G/F motif is dispensable for some of DnaJ's functions. Moreover, the fact that the G/F motif is always found immediately downstream of the J-domain has led to the suggestion that it may play an auxiliary role in modulating DnaK's chaperone activity, synergistic with the J-domain (Wall et al., 1994).


Figure 1: Alignment of the G/F motif present in most DnaJ family members. Sequences homologous to E. coli DnaJ were obtained using the TFastA search program (Pearson and Lipman, 1988) of the GenEMBL data base. Sequences were aligned using the Genetics Computer Group (GCG) program PileUp (gap weight, 3.0; gap length, 0.1). A consensus sequence was obtained with the Pretty program (plurality, 7; threshold, 1.0). Residues that contributed toward the consensus are capitalized, while residues that are identical in more than 80% of the sequences are indicted as white letters with a black background. An abbreviation of the organism's or proteins (yeast) name is given in the left margin. The G/F residues, deleted in the DnaJDelta77-87 and DnaJDelta77-107 constructs, are indicated by black bars.



To address what role the G/F motif plays in DnaJ function, we constructed two DnaJ internal deletion mutants. The first, DnaJDelta77-87, has the first 11 amino acids deleted, 9 of which are Gly residues (Fig. 1). The highly conserved DI(V)F residues are not deleted in this construct. In contrast, DnaJDelta77-107 has the entire 31 amino acid G/F motif deleted (Fig. 1, see ``Materials and Methods''). The genes that code for these mutants are carried on the pTTQ19 plasmid (Amersham Corp.), under the tight transcriptional control of the lacI^q gene product, whose gene is simultaneously carried on the plasmid. To test for functional activity, these plasmids were transformed into E. coli strains that have been deleted for the dnaJ gene. Interestingly, the DnaJDelta77-87 mutant complemented all bacterial and bacteriophage growth defects associated with the dnaJ null mutants to near wild type levels at either 30 or 42 °C. In contrast, the DnaJDelta77-107 mutant did not complement any of the bacterial growth defects associated with the DnaJ null mutant. In fact, cells harboring the DnaJDelta77-107 allele grew considerably slower than the isogenic bacterial strains that carried the parental vector plasmid, suggesting that the slightly overproduced (leq2-fold) DnaJDelta77-107 was somehow poisonous to cell growth, perhaps through nonproductive interactions with DnaK (see below).

The DnaJDelta77-107 protein was able to complement bacteriophage growth, albeit much less efficiently (50-fold lower bacteriophage yield) than the DnaJ protein in the same background, demonstrating that at least some of its functions were retained. Bacteriophage grew to a similar extent at either 30 or 42 °C, showing that DnaJDelta77-107 does not exhibit a temperature-sensitive phenotype for growth per se as other dnaJ mutants do (Wall et al., 1994), but simply works less efficiently at all temperatures compared to DnaJ. On the basis of these in vivo results, we decided to biochemically characterize in detail the DnaJDelta77-107 mutant protein, not only because it exhibited interesting phenotypes, but also because the DnaJDelta77-87 protein appeared to have identical activities as DnaJ.

DnaJDelta77-107 Interacts with DnaK

It has previously been shown that DnaJ stimulates the hydrolysis of DnaK-bound ATP (Liberek et al., 1991a) and that the amino-terminal 108 amino acids of DnaJ are both necessary and sufficient for such a function (Wall et al., 1994). To further delineate the role, if any, that the G/F motif plays in these processes, we tested the ability of purified DnaJDelta77-107 to stimulate DnaK's ATPase activity. Fig. 2shows that DnaJDelta77-107 stimulates DnaK's activity to the same extent as DnaJ does. This result is consistent with our earlier conclusion that the highly conserved J-domain is responsible for stimulating DnaK's ATPase activity (Wall et al., 1994).


Figure 2: Interactions of various DnaJ proteins with DnaK. A, titration of the ability of DnaJ proteins to stimulate DnaK's ATPase activity. The reaction mixtures contained ATP (200 µM), DnaK (0.41 µM), GrpE (0.90 µM), and varing amounts of DnaJ proteins, as described (Liberek et al., 1991a). B, ability of DnaJ proteins to bind DnaK. The indicated DnaJ proteins (0.5 µg/well) or BSA (100 µg/well) were added to microtiter plate wells and treated as described under ``Materials and Methods.'' Following a series of blocking and washing steps (see ``Materials and Methods''), various amounts of DnaK were added for 45 min at room temperature. Subsequently, the amount of bound DnaK protein was measured by ELISA using rabbit antiserum against DnaK (1:7,000 dilution) as described under ``Materials and Methods.''



Our earlier work established that a His Gln substitution at amino acid 33 (DnaJ259) resulted in a protein that was completely defective in stimulating DnaK's ATPase activity (Wall et al., 1994). However, this result did not distinguish between a possible defect in the physical interaction between DnaK and DnaJ259, or alternatively a potential catalytic defect in DnaJ259. To establish what role the J-domain and G/F motifs play in DnaJ's physical interaction with DnaK, we tested the ability of DnaJ259 and DnaJDelta77-107 mutant proteins to bind DnaK. To assay for this protein-protein interaction we used a modified ELISA approach (see ``Materials and Methods''), since it was recently shown to be a sensitive assay to monitor DnaK-DnaJ interactions. (^3)Fig. 2B shows that in the presence of ATP DnaJDelta77-107 exhibits a high affinity toward DnaK, nearly identical to that of wild type DnaJ. In contrast, DnaJ259 was severely defective in its ability to bind DnaK (Fig. 2B). These results are in good overall agreement with the ATPase stimulation data. In addition, as judged by partial trypsin digestion analysis, DnaJDelta77-107 was found to regulate DnaK's conformational state in an ATP-dependent reaction similar to DnaJ (Wall et al., 1994; data not shown). Therefore, we conclude that the J-domain plays the major role in determining the biochemical affinity of DnaJ to DnaK.

DnaJDelta77-107 Is Defective in Activating DnaK to Bind

Based on the fact that DnaJ12 could activate DnaK to bind and could also down-regulate the heat-shock response in vivo, while DnaJDelta77-107 and DnaJ259 could not down-regulate the heat-shock response (see below),^1 we tested the ability of these mutant proteins to bind and to activate DnaK to bind in the presence of ATP. As expected, DnaJDelta77-107 and DnaJ259 possess similar affinities toward as DnaJ (Fig. 3A). In contrast, in the presence of ATP DnaK did not significantly interact with (Fig. 3B), under the buffer conditions used, which includes 0.1% Triton X-100. However, if wild type DnaJ was added to the reaction, in the presence of ATP, DnaK was able to bind (Fig. 3B). This stimulatory role of DnaJ can be titrated and reaches saturation at a molar ratio of 1 DnaJ (dimer) to 10 DnaK (monomers). These results suggest that DnaJ can catalytically activate DnaK to bind , in the presence of ATP, but not in its absence. This activating role of DnaJ was substrate specific, since DnaJ did not activate DnaK to bind BSA (Fig. 3B).


Figure 3: Ability of various DnaJ proteins to bind and to activate DnaK to bind . A, the indicated DnaJ proteins (0.5 µg/well) or BSA (100 µg/well) were incubated with various amounts of as described under ``Materials and Methods.'' The amount of bound protein was measured by ELISA using rabbit antiserum against (1:2,000 dilution) as described under ``Materials and Methods.'' B, protein (0.5 µg/well) or BSA (100 µg/well) were incubated with DnaK (80 ng), ATP (1 mM) and various amounts of DnaJ proteins. The amount of DnaK bound to the or BSA wells was estimated by ELISA using antiserum to DnaK (1:7,000 dilution) as described under ``Materials and Methods.''



In parallel experiments, we confirmed and extended our earlier results, demonstrating that DnaJ259 was completely defective in activating DnaK to bind in an ATP-dependent reaction by a factor of at least 100. Interestingly, DnaJDelta77-107 was severely defective in its ability to activate DnaK to bind (Fig. 3B), although it exhibited a weak, yet reproducible stimulatory activity. In analogous experiments in which firefly luciferase was substituted for , a protein known to interact with DnaK and DnaJ (Schröder et al., 1993; Wall, 1994), DnaJDelta77-107 was again found to be defective in activating DnaK to bind luciferase in an ATP-dependent reaction as compared to DnaJ (Wall, 1994).

In a second approach, glycerol gradient sedimentation also showed that DnaJDelta77-107 was unable to activate DnaK to bind in the presence of ATP (not shown). These results indicate that the stimulatory role DnaJ plays in DnaK's ATPase activity is not sufficient by itself to activate DnaK to bind to at least some of its substrates. Thus, we conclude that the G/F motif plays an auxiliary, yet important, role in activating DnaK to bind substrates that is clearly separable from the stimulatory role that DnaJ plays in stimulating DnaK's ATPase activity.

DnaJ12, But Not DnaJ259 or DnaJDelta77-107 Can Negatively Regulate E. coli's Heat-shock Response

Mutations in the dnaJ gene have been shown previously to result in the overexpression of heat-shock genes, even under non-heat-shock conditions (Sell et al., 1990; Straus et al., 1990). This phenotype is correlated with the in vivo stabilization of the major E. coli heat-shock transcriptional regulator, , an extremely unstable protein, with a half-life of 1 min at 30 °C (Straus et al., 1990; Straus et al., 1987). Mutations in either dnaK or grpE also result in a similar phenotype (Straus et al., 1990; Tilly et al., 1983). These observations form the basis for the conclusion that these three molecular chaperones are involved in the negative autoregulation of the heat-shock response (for reviews, see Craig and Gross, 1991; Yura et al., 1993). This conclusion has been further substantiated by the finding that DnaK and DnaJ together or separately physically interact with (Gamer et al., 1992; Liberek et al., 1992; Liberek and Georgopoulos, 1993). Specifically, DnaJ exhibits a high affinity for , while DnaK exhibits a much lower affinity. However, as discussed, in the presence of ATP and DnaJ, DnaK can be activated to bind with high affinity (Liberek and Georgopoulos, 1993). Interestingly, DnaJ12, which does not bind because its substrate binding domain has been deleted, is still capable of activating DnaK to bind . Conversely, DnaJ259 binds but does not activate DnaK to bind .^1 Previously it has been known that the dnaJ259 mutation results in the overproduction of the heat-shock proteins (Sell et al., 1990; Straus et al., 1990). Thus, the demonstrated binding of DnaJ259 to is not sufficient to either destabilize in vivo, or to inhibit its function, thus resulting in the turn off the heat-shock response.

To test whether or not the important biochemical interaction in vivo for heat-shock autoregulation was the DnaK- interaction, we tested the ability of the dnaJ12 and dnaJDelta77-107 mutant alleles to down-regulate the heat-shock response, as judged by the expression from a heat-shock reporter gene fusion, groE::lacZ. Using isogenic bacteria we found that the dnaJ12 allele could down-regulate the overproduction of the heat-shock response caused by a dnaJ null mutation, while dnaJDelta77-107 could not (Fig. 4). As controls, the parental vector alone or the dnaJ259-bearing plasmid could not down-regulate the expression of the groE::lacZ reporter gene, while an isogenic dnaJ-containing plasmid could (Fig. 4). Consistent with our other results, the dnaJDelta77-87-containing plasmid was able to down-regulate the expression of the groE::lacZ reporter gene (Fig. 4). These results are highly reproducible since they were obtained with two independently transformed strains, and six independent beta-galactosidase assays gave similar results.


Figure 4: Ability of various DnaJ proteins to down regulate E. coli's heat-shock response. A dnaJ mutant strain, that contains the heat-shock reporter gene ØpgroE-lacZ integrated into the chromosome, was transformed with the indicated plasmid-borne dnaJ alleles or the plasmid vector. The beta-galactosidase activity (Miller units) of these isogenic bacteria was then determined at 30 °C as described previously (Miller, 1992). The values given are an average of six independent determinations.



To further substantiate these results, the same plasmids were transformed into a dnaJ mutant strain, carrying a different heat-shock reporter gene fusion lon::lacZ (Missiakas et al., 1993). Again, DnaJ12 was found to down-regulate transcription from this heat-shock reporter fusion to a level similar to that seen with isogenic dnaJ bacteria, while the dnaJ259- and dnaJDelta77-107-bearing isogenic plasmid constructs did not (data not shown). Together with our in vitro data, we conclude that it is the DnaJ-catalyzed ability of DnaK to recognize and bind that is critical to the down-regulation of the heat-shock response in E. coli and not the ability of DnaJ to bind , as has been previously suggested by others (Bukau, 1993). As previously mentioned, the DnaJ protein levels produced from our expression plasmid vectors are no more than 2-fold higher than those found for wild type bacteria expressing DnaJ from a single copy gene, as judged by quantitative Western analysis (not shown).


CONCLUSIONS

A central question in the protein chaperone field is how DnaK and its eukaryotic counterparts bind and release substrates. Increasing evidence indicates that in vivo substrate binding by DnaK is often a coordinated process with DnaJ and ATP (Langer et al., 1992; Liberek and Georgopoulos, 1993; Osipiuk et al., 1993; Wickner et al., 1991; Zylicz, 1993). We have shown here and elsewhere that the critical steps in this concerted process involve the ability of DnaJ to stimulate ATP hydrolysis by DnaK, resulting in a large conformational change in DnaK (Liberek et al., 1991a; Wall et al., 1994). These steps are required for ATP-dependent binding of DnaK to some of its substrates, since the DnaJ259 mutant protein does not possess these activities and does not activate DnaK to bind such substrates, while DnaJ12 has these activities and stimulates DnaK to bind .^1

In this work we have further investigated this important activation process by deleting the conserved G/F motif (module), which is present in the DnaJ12 mutant, while leaving the highly conserved J-domain and the rest of the DnaJ protein intact. The resulting DnaJDelta77-107 mutant protein contains a 31-amino-acid internal deletion substituted with a 6 amino acid insertion. DnaJDelta77-107 was found to stimulate DnaK's ATPase activity like wild type DnaJ, but, surprisingly, it does not activate DnaK to bind . In this respect it is interesting that Schmid et al.(1994) recently showed that DnaK has a significantly faster on rate for substrates when bound to ATP. However, this increased on rate for substrates is compensated by an even faster off rate in the presence of ATP, thus the net result is that DnaK exhibits an approximately 10-fold lower affinity toward substrates in the presence of ATP, as previously established (Flynn et al., 1989; Liberek et al., 1991b).

In agreement with biophysical and biochemical data, we interpret the process of ATP binding to DnaK as an ``activation'' step, in which the substrate binding domain/pocket of DnaK becomes more exposed to the solvent (Buchberger et al., 1994; Liberek et al., 1991b; Palleros et al., 1991; Schmid et al., 1994). Although substrates may bind faster in this conformation, they are also released at a fast rate (Schmid et al., 1994). In our model, the role of DnaJ is to both help deliver substrates to this substrate-binding pocket of DnaK and to stimulate pocket closure (i.e. conformational change), presumably through ATP hydrolysis. This regulated reaction ``locks'' DnaK into a conformation that stably binds substrates, i.e. slow off rate in the ADP form (Palleros et al., 1991; Schmid et al., 1994). This model is consistent with our findings that DnaJ ``triggers'' DnaK into the presumed ``closed'' conformation in the presence of ATP (Wall et al., 1994).

One interpretation of the failure of DnaJDelta77-107 to activate DnaK to bind is that although DnaJDelta77-107 stimulates ATP hydrolysis and DnaK ``closure,'' it fails to ``lock'' DnaK into a conformation that stably binds . This could be explained mechanistically, for example, by a failure of DnaJDelta77-107 to regulate a possible phosphorylation or oligomeric state of DnaK, which has been suggested to modulate DnaK's affinity toward substrates (Schmid et al., 1994; Sherman and Goldberg, 1993). (^4)Alternatively, the DnaJDelta77-107 mutant protein could result in a conformational perturbation such that it can no longer activate DnaK to bind substrates. However, since DnaJDelta77-107 can both stimulate DnaK's ATPase activity and bind to substrates at wild type efficiencies argues against DnaJDelta77-107 being dramatically misfolded. Along these lines, the failure of DnaJDelta77-107 to activate DnaK to bind substrates could result in a structural defect such that it cannot simultaneously orchestrate substrate presentation and ``activation'' to DnaK. In this interpretation, the G/F module would serve as a linker or hinge between domains of DnaJ (Silver and Way, 1993), thus allowing both domains to be properly oriented when interacting with DnaK. In either interpretation, we conclude that the G/F motif of DnaJ is serving an ``auxiliary,'' yet important regulatory role in activating DnaK to bind substrates (see below).

Interestingly, some DnaJ family members do not contain the G/F motif (Bork et al., 1992). For example Sec63, which is a Yeast homolog and is a component of the protein transport machinery into the endoplasmic reticulum, has a J-domain localized in the lumen of the endoplasmic reticulum (reviewed in Sanders and Schekman, 1992). It is thought that this domain interacts with the ER-localized BiP (Hsp70 homolog) in protein transport and folding (Feldheim et al., 1992; Sanders and Schekman, 1992). We suspect that the coordinated Sec63-BiP complex performs different biochemical functions than, for instance, the binding of E. coli's DnaK/DnaJ to or to the ``O-some'' complex in DNA replication (Zylicz, 1993). Most likely, the Sec63-BiP machinery rapidly binds and releases unfolded/extended polypeptides (Blond-Elguindi et al., 1993; Flynn et al., 1991) as they are traversing the ER membrane, thus facilitating protein transport and folding (Sanders and Schekman, 1992). Thus, in this scheme Sec63 does not possess the G/F motif, simply because it does not require the ``locking'' or ``stabilizing'' functions of DnaJ. In the examples of and P, the DnaK/DnaJ machinery binds to ``native proteins'' (i.e. already in a biologically active form) and thus, most likely, not present in an unfolded/extended state. Moreover, such DnaK/DnaJ/substrate complexes may be longer lived, especially because ATP hydrolysis ensures their stable association. Consistent with this, Brodsky et al. (1993) found that Hsc70, Ssa1 (the DnaK eukaryotic homologs), as well as E. coli's DnaK protein could not substitute for BiP function in a reconstituted protein transport system. Conversely, BiP did not substitute for the function of cytosolic Hsc70, required for proper protein transport on the cytosolic side of the ER membrane. Thus, although these DnaK-like proteins are functioning in an overall similar manner to carry out similar biological tasks, nevertheless, they are not interchangeable.

Recently, additional DnaJ and DnaK family members have been discovered in E. coli (Kawula and Lelivelt, 1994; Seaton and Vickery, 1994; Ueguchi et al., 1994; Yura et al., 1992), bringing the total number of DnaJ family members to four and DnaK family members to two. Interestingly, all other DnaJ family members do not contain the G/F motif. This may reflect more specialized roles for these putative DnaJ protein chaperones. Nevertheless, genetic evidence indicates that at least one of these DnaJ family members can functionally substitute for the ``classical'' DnaJ protein (Ueguchi et al., 1994). For example, multicopy levels of the cbpA homolog gene restores bacterial growth at high temperatures (Ueguchi et al., 1994), (^5)as well as bacteriophage growth to a dnaJ strain (Ueguchi et al., 1994). These results are also consistent with our findings presented here, namely that multicopy levels of dnaJDelta77-107 support growth. The discovery of the cbpA gene of E. coli helps explain the higher basal level of DNA replication activity found in a crude extract system prepared from a dnaJ strain compared to a dnaJ259 strain (Wall et al., 1994).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM23917, Swiss National Foundation Grant 31-31129.91, the Canton of Geneva, and Grant P303 042 06 from the Polish State Committee for Scientific Research. 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: Dept. of Biochemistry, Stanford University, Stanford, CA 94305. Tel.: 415-723-5685; Fax: 415-723-6783; dwall{at}cmgm.stanford.edu.

(^1)
K. Liberek, D. Wall, and C. Georgopoulos, manuscript in preparation.

(^2)
The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin.

(^3)
A. Wawrzynow and M. Zylicz, unpublished data.

(^4)
R. McMacken, personal communication.

(^5)
D. Wall and C. Georgopoulos, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. K. Liberek and A. Wawrzynow for sharing unpublished data.


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