Proc. NatI. Acad. Sci. USA Vol. 88, pp. 2874-2878, April 1991 Biochemistry Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK KRZYSZTOF LIBEREK*t, JAROSLAW MARSZALEK*, DEBBIE ANGt, COSTA GEORGOPOULOStt, AND MACIEJ ZYLICZ* *Division of Biophysics, Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822, Gdansk, Poland; and tDepartment of Cellular, Viral, and Molecular Biology, University of Utah School of Medicine, Salt Lake City, UT 84132 Communicated by Allan M. Campbell, December 31, 1990

ABSTRACT The products of the Escherichia coli dnaK, when ATP was added to complexes of hsc70 (a constitutive dnaJ, and grpE heat shock have been previously shown member of the family) and p53 (an anti-oncogenic to be essential for A DNA replication at all protein) (9), immunoglobulin heavy chains and their binding temperatures and for bacterial survival under certain condi- protein BiP (10), and uncoating ATPase complexed with tions. DnaK, the bacterial hsp7O analogue clathrin or membrane vesicles (11, 12). Recently, Beckmann and putative chaperonin, possesses a weak ATPase activity. et al. (13) have shown that the cytosolic hsp70 proteins may Previous work has shown that ATP hydrolysis allows the interact with a large number of newly synthesized proteins. release ofvarious polypeptides complexed with DnaK. Here we These examples suggest that ATP-dependent release ofhsp70 demonstrate that the ATPase activity of DnaK can be greatly from a complex with its substrate is a common feature of the stimulated, up to 50-fold, in the simultaneous presence of the hsp70 family. However, in all the described cases, the DnaJ and GrpE heat shock proteins. The presence of either detected rate of ATP hydrolysis catalyzed by DnaK, hsp70, DnaJ or GrpE alone results in a slight stimulation of the or BiP is relatively low, approximately 0.1-1.0 molecule of ATPase activity of DnaK. The action of the DnaJ and GrpE ATP per min per monomer. Flynn et al. (14) have recently proteins may be sequential, since the presence of DnaJ alone shown that two members of the hsp70 family of proteins, leads to an acceleration in the rate of hydrolysis of the hsc70 and BiP, can bind short peptides (8-25 residues) with DnaK-bound ATP. The presence of GrpE alone increases the no apparent specificity. Such binding leads to a 4-fold stim- rate of release of bound ATP or ADP without affecting the rate ulation of the ATPase activity of hsp70, resulting in the of hydrolysis. The stimulation of the ATPase activity of DnaK release of the bound peptides (14). may contribute to its more efficient recycling, and it helps In the case of bacteriophage A DNA replication in E. coli, explaln why mutations in dnaK, dnaJ, or grpE genes often the DnaK protein, in conjunction with another heat shock exhibit similar pleiotropic phenotypes. protein, DnaJ, is responsible for the partial disassembly of the preprimosomal complex. ATP-dependent release of the The Escherichia coli dnaK product, the prokaryotic hydrophobic AP replication protein from the preprimosomal analogue ofhsp70, the eukaryotic 70-kDa heat shock protein, complex, located at oriA, triggers the initiation of A DNA participates in a variety of basic cellular functions: (i) sur- replication by allowing the DnaB helicase to unwind the vival of under different stress conditions, (ii) initi- duplex template near oriA (15, 16). The release of AP protein ation of bacteriophage A and E. coli oriC-dependent DNA from the complex is dependent on ATP hydrolysis, presum- replication, (iii) regulation of cell division, (iv) modulation of ably catalyzed by the DnaK protein (17). In the in vitro proteolysis, (v) protein phosphorylation, and (vi) transport of replication system, DnaK protein is required at a concentra- proteins across membranes (reviewed in refs. 1-3). Such a tion 20-fold higher relative to the other replication proteins. broad spectrum of action suggests involvement of the DnaK However, in the presence of another heat shock protein, protein in some general mechanisms crucial for the survival GrpE (18), the requirement for DnaK protein drops 10-fold of the cell. Pelham (4) has suggested that the heat shock (16). The stimulation ofDnaK's ATPase activity by DnaJ and proteins belonging to the hsp70 family are involved in binding GrpE may lead to a more efficient release of DnaK-bound to the hydrophobic domains ofother proteins, exposed either polypeptides such as AP, thus aiding the intracellular recy- naturally or as a result of stressful conditions. Such binding cling of the DnaK protein. and release, following ATP hydrolysis, may allow the disas- sembly of "dead-end" protein structures formed under stress MATERIALS AND METHODS conditions. In support of this hypothesis, we have recently shown that the DnaK protein protects E. coli RNA polymer- Bacteria and Plasmids. The bacterial strains, as well as the ase from heat inactivation by preventing its aggregation. In various plasmids used in the course of this work, have been addition, in an ATP-dependent reaction, the DnaK protein described previously (16, 18-22). can also dissolve the RNA polymerase aggregates formed at Proteins. Highly purified proteins (90% or greater purity) high temperature, leading to a complete restoration of RNA were used. Their specific activities were as follows: DnaK, 3 polymerase activity (5). Early evidence that ATP may be x 103 units per mg of protein (21); DnaJ, 4 x 105 units per mg involved in hsp70 function was the observation that the E. of protein [purified as described in ref. 22 with the modifi- coli DnaK protein has a weak ATPase activity (6). It was cation described by Zylicz et al. (16)]; and GrpE, 5 x 105 units subsequently shown that members of the mammalian hsp70 per mg of protein (21). A unit of activity catalyzes the family of proteins bind tightly to ATP cross-linked to an incorporation of 1 pmol of deoxynucleotides per min into agarose matrix (7) and that ATP is required for release of trichloroacetic acid-insoluble material under standard Adv hsp70 protein from nuclei (8). Similar results were obtained DNA replication assay conditions. Complementation of the Adv DNA replication assay for DnaK, DnaJ, and GrpE has been described (20-22). The temperature- The publication costs of this article were defrayed in part by page charge activities payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2874 Downloaded by guest on September 28, 2021 Biochemistry: Liberek et al. Proc. Natl. Acad. Sci. USA 88 (1991) 2875 sensitive proteins encoded by dnaK756 (21) and grpE280 (23) The availability ofpurified DnaJ and GrpE proteins, which were purified as previously described. are required at the same step in A DNA replication as DnaK ATPase Assay and Kinetic Analysis. DnaK ATPase activity (15, 16), allowed us to test whether the ATPase activity of was determined from the amount of [32p]- or [3H]ADP DnaK is modulated by these two heat shock proteins (18, 26). produced from [y-32P]ATP (0.01 /Ci with a specific activity The addition of either GrpE or DnaJ protein alone to the of3000 Ci/mmol; NEN/Du Pont; 1 Ci = 37 GBq) or [3H]ATP DnaK ATPase assay had a small but reproducible effect (no (0.5 ,tCi with a specific activity of 30 Ci/mmol; [2,8-3H]ATP more than 2-fold) on the rate of ATP hydrolysis catalyzed by from ICN), respectively, at 30°C. DnaK protein (Fig. 1). However, when both GrpE and DnaJ The reaction mixture contained 30 mM 4-(2 hydroxyme- were present, the rate of ATP hydrolysis by DnaK increased thyl)-1-piperazineethanesulfonic acid (Hepes) buffer at pH approximately 20-fold relative to DnaK protein alone (Fig. 1). 7.6, 40 mM KCI, 50 mM NaCI, 7 mM magnesium acetate, 2 In control experiments, it was shown that neither DnaJ alone, mM dithiothreitol, bovine serum albumin at 0.29 mg/ml, GrpE alone, nor DnaJ and GrpE together display any ATPase ATP, and other compounds in the amounts indicated in the activity in the absence of DnaK (Fig. 1). The extent of figures. stimulation of DnaK's ATPase activity by DnaJ and GrpE The reaction was initiated by the addition ofATP to a 25-,ul was somewhat variable, ranging from 5- to 50-fold, depending assay mixture containing the various proteins. At different on the particular preparations of the three proteins. time intervals thereafter, 2-,ul samples from the incubation To test whether the presence of GrpE and DnaJ changes mixture were applied directly onto polyethyleneimine (PEI)- DnaK's affinity for ATP and/or increases the maximal ve- cellulose thin-layer sheets. The substrate and products ofthe locity (Vmax) of the ATPase reaction, we measured the initial reaction were separated by one-dimensional chromatography velocity of the reaction as a function of ATP concentration. using 1 M formic acid/1 M LiCl (1:1, vol/vol) (24). The ATP The Michaelis-Menten curves presented in Fig. 2A clearly and ADP spots were identified by chromatography with show that the Vma,, of ATPase activity increases significantly unlabeled standards and cut out, and the radioactivity was in the presence of DnaJ and GrpE proteins, from approxi- determined by liquid scintillation counting. mately 3.5 nmol to 90 nmol of ATP hydrolyzed per min per The initial velocity was calculated from the linear part of mg of DnaK. The Km of DnaK's ATPase reaction was the reaction product concentration vs. time plot by the linear estimated to be approximately 20 ,uM. This value was ap- regression method. proximately the same with or without DnaJ and/or GrpE. Separation of DnaK-Bound Nucleotides. A Bio-Rad P-60 Because the in vivo physiological concentration of ATP is in column (0.5 x 7 cm) was equilibrated with buffer A: 50 mM the range of 2-4 mM, the effect of DnaJ and GrpE proteins on the Vmax of DnaK's ATPase activity could be especially Tris HCI, pH 7.8/50 mM NaCl/50 mM KCl/10 mM MgCl2/2 important. mM dithiothreitol. The 50-,l reaction mixture, containing 125 Titration of GrpE or DnaJ protein in the DnaK ATPase ,ug of DnaK and 100 ,uM ATP (10 ,uCi of [a-32P]ATP; assay showed that components in the ratio of 1 mol of DnaK NEN/Du Pont) in buffer A, was incubated for 5 min at 30°C to 1-2 mol of DnaJ to 1-2 mol of GrpE substantially stimu- to allow the formation of a DnaK-ATP complex. Following lated the activity (Fig. 3). To verify that the enhancement of this, the mixture was loaded onto the P-60 column at room the ATPase activity is due to the DnaK protein, we purified temperature. Approximately 100-,ul fractions were collected the temperature-sensitive protein encoded by the dnaK756 and analyzed directly by liquid scintillation counting. The mutant gene and examined its ATPase activity in the pres- void volume fraction, containing the DnaK protein (com- ence and absence of the DnaJ and GrpE proteins. Surpris- plexed with nucleotide) asjudged by Coomassie blue staining ingly, we found that the DnaK756 mutant protein, when after SDS/PAGE, was quickly frozen in liquid nitrogen and purified on an ATP-agarose column in a manner similar to used in further experiments. The concentration of DnaK wild-type DnaK, possesses a relatively high ATPase activity protein was 1 ug/dul (88,000 cpm per ,ug of DnaK). (50-fold higher than wild type), even in the absence of DnaJ To determine the effect of the DnaJ and GrpE proteins on and GrpE. The addition of DnaJ and GrpE (at the concen- the DnaK-bound nucleotides, aliquots containing 2.2 ILM tration optimal for wild-type DnaK's ATPase activity) stim- DnaK protein complexed with nucleotide were incubated (i) ulated this activity only 2- to 3-fold (Fig. 2B). Previously we without any additional protein, (ii) with DnaJ (3.1 ,uM), (iii) with GrpE (2.4 ,uM), or (iv) with both DnaJ (3.1 uM) and GrpE (2.4 ,uM), in a total reaction volume of 40 ,ul in buffer A. The incubation was carried out at 30°C for 1 min. Fol- lowing this, the reaction mixtures were loaded on a small Bio-Rad P-60 column (0.4 x 4 cm), equilibrated with buffer -6 201- A, and approximately 100-,lI fractions were collected. The 0 presence of radioactivity in each fraction was determined by liquid scintillation counting. The presence of [a-32P]ATP and [a-32P]ADP was determined by thin-layer chromatography -Ca- on PEI-cellulose (24) and autoradiography. For quantitation, the areas corresponding to ATP and ADP were excised and subjected to liquid scintillation counting.

j RESULTS I I -d ^ nf% The DNA-independent ATPase activity of DnaK protein is U 10 zu very low (15 nmol of ATP hydrolyzed per min per mg of Time, min protein at 30°C, pH 8.8), giving a turnover rate of 1 molecule FIG. 1. Kinetics of ATP hydrolysis. The 25-,lI incubation mix- of ATP hydrolyzed per min per molecule of DnaK. Under tures contained 0.46 ,uM DnaK, 0.8 ,uM DnaJ, or 0.25 ,uM GrpE. The more physiological conditions (pH 7.0-7.6), the rate is ap- concentration of ATP was 100 ,uM. The rest of the experimental proximately 1/4th of this (6). None of the other ribo- or conditions and details are described in Materials and Methods. o, deoxyribonucleotides tested were found to be better sub- DnaK alone; *, DnaK and GrpE; * DnaK and DnaJ; 0. DnaK, strates for this enzyme (25). DnaJ, and GrpE; *, DnaJ and GrpE. Downloaded by guest on September 28, 2021 2876 Biochemistry: Liberek et al. Proc. Natl. Acad. Sci. USA 88 (1991)

A

100- * * I a ._ 40 Q.

C 20- 0) E E 0 0) I Q 0 I I0 0.2 0.4 0.6 0.8 1.0 E E.0 c 0 DnaJ protein, ,uM ? 600 E B c C6 c6 300 2 is 3 400 i_ 200 300*

200-

100'

0.0 1.0 ., I 0.2 0.4 0.6 0.8 0.4 0.6 0.8 ATP, mM GrpE protein, uM FIG. 2. Substrate saturation experiments. (A) Reaction mixture of DnaK (1.7 ,uM) alone (*), and DnaK (0.17 1AM) in the presence of FIG. 3. Titration of DnaJ and GrpE protein requirement. (A) DnaJ (0.2 ,uM) and GrpE (0.17 uM) .(o). (B) DnaK756 (17 nM) alone Reaction mixture of DnaK (0.57 jLM) and GrpE (0.83 4M) was incubated with various amounts of and (*) and in the presence of flnaJ (30 nM) and GrpE (33 nM) (o). DnaJ. (B) DnaK (0.57 tuM) DnaJ (0.7 ,tM) were incubated with various concentrations ofGrpE. had shown that the DnaK756 protein possesses a somewhat In all assays the concentration of ATP was 100 AM. weaker and more heat-labile ATPase activity than wild-type activity, we first isolated a complex between DnaK and DnaK. This result was obtained after immunoprecipitation substrate nucleotides. This was achieved by incubating and treatment for 5 min at 950C (6). To ensure the reproduc- DnaK with [a-32P]ATP for a short time and isolating the ibility ofDnaK756's high ATPase activity, three independent protein-nucleotide complex by size chromatography (see preparations were made; all were shown to possess equiva- lent high levels of ATPase activity (data not shown). Materials and Methods). We estimate that more than 60% of Effects of Temperature. All the proteins used in our exper- iments belong to the family of heat shock proteins, which are involved in cellular metabolism under stress conditions, such 150- as high temperature. Therefore, we determined the initial E velocity of DnaK's ATPase activity as a function of temper- ature. It was shown previously that DnaK possesses an unusually heat-stable ATPase activity (6). We observed that the maximal initial velocity ofATP hydrolysis by DnaK alone X 100 E was within the 50-550C range (reaching a 4-fold higher value C at 52TC than at 300C; Fig. 4). In the presence ofboth DnaJ and GrpE, DnaK's ATPase activity was efficiently stimulated; however, the ratio of its activities in the presence and absence of DnaJ and GrpE remained the same up to 520C. GrpE280 Mutant Protein Is Inactive. We recently purified the mutant GrpE280 protein and showed that, like the wild- 30 r type protein, it binds to DnaK. However, unlike the wild- type protein, it rapidly dissociates from this complex (23). We found that the GrpE280 mutant protein, in the presence or 30 40 50 60 absence of DnaJ, does not significantly stimulate DnaK's Temperature, OC ATPase activity (data not shown). We conclude that a "stable" interaction between the DnaK is FIG. 4. Influence of temperature on DnaK's rate of ATP hydrol- and GrpE proteins ysis. Both the appropriate protein mixture and the ATP-containing needed for successful stimulation of DnaK's ATPase activ- reaction mixture were incubated separately for 2 min at the indicated ity. reaction temperature. The reaction was started by the addition ofthe a Mode of Action of DnaJ and GrpE. In search for the protein mixture to the reaction mixture. n, DnaK (0.17 ,uM) alone; molecular mechanism of the stimulation of DnaK's ATPase *, DnaK (0.17 gM), DnaJ (0.2 gM), and GrpE (0.25 /AM). Downloaded by guest on September 28, 2021 Biochemistry: Liberek et al. Proc. Natl. Acad. Sci. USA 88 (1991) 2877 the DnaK protein is complexed with nucleotide. The type of ase, as well as the eukaryotic protein p53 (5, 6, 17, 22, 27). nucleotide bound to DnaK was determined by thin-layer Previous work has shown that DnaK, when present at high chromatography. In a typical experiment, approximately concentrations, releases APfrom the preprimosomal complex 60% of the bound nucleotide is ADP and 40 is ATP (Fig. 5, in an ATP-dependent reaction only in the presence of DnaJ lane a). Gel-filtration experiments using the isolated complex (15-17). This slow reaction is much more efficient (at least showed that the bound ADP is preferentially released from 10-fold) when DnaJ and GrpE are simultaneously present DnaK during the course of the reaction (Fig. 5A, approxi- (16). These two experimental facts suggested to us that the mately fractions 4-8), although neither ADP nor ATP is rate of ATP-dependent release of DnaK from its bound released efficiently-i.e., the majority of ATP and ADP substrate could be accelerated in the presence of DnaJ and remain bound to DnaK (Fig. 5A, fractions 2 and 3). When the GrpE. We found that the ATPase activity of DnaK is effi- DnaK-nucleotide complex is incubated with DnaJ for 1 min at ciently stimulated by the presence of both DnaJ and GrpE. 300C, the bound ATP is very efficiently converted to ADP The extent of stimulation was variable, between 5- and (Fig. SB; lane b). However, gel filtration shows that the 50-fold, depending on the particular protein preparations presence of DnaJ does not significantly stimulate the release used. We do not know the reason for this variability. How- of the bound ADP. ever, in all cases examined, each DnaJ or GrpE preparation The addition of GrpE alone for 1 min at 30TC does not alone did not affect DnaK's ATPase activity by more than stimulate the hydrolysis of either ATP or ADP. Rather, it 2-fold. Similarly, the addition of heat-denatured AO or heat- results in the efficient release of both ATP and ADP from denatured AP, bovine serum albumin, or unfolded bovine DnaK (Fig. 5C). As expected, the simultaneous addition of pancreatic trypsin inhibitor did not appreciably stimulate both DnaJ and GrpE results in both the efficient hydrolysis DnaK's ATPase activity. Hence, the stimulation of the of ATP to ADP and the efficient release of bound ATP/ADP ATPase reaction requires the joint presence of DnaJ and (Fig. 5D). GrpE. The Vm., but not the Km, of DnaK's ATPase activity Addition of unfolded bovine pancreatic trypsin inhibitor was appreciably affected by the presence of DnaJ and GrpE. which binds tightly to DnaK (unpublished data), does not Since the Km of the ATPase reaction was estimated to be stimulate either the hydrolysis of bound ATP to ADP or the approximately 20 ,uM, and the intracellular concentration of release of the DnaK-bound nucleotides (data not shown). As ATP is in the range of 2-4 mM, stimulation ofDnaK by DnaJ an additional control, we showed that purified GrpE280 and GrpE could have important biological consequences. mutant had The exact role that DnaJ and GrpE play in the stimulation protein similarly no discernible effect on the of DnaK's ATPase activity was investigated by taking advan- hydrolysis or release of the DnaK-bound nucleotides (data tage ofthe fact that ATP hydrolysis is slow (approximately one not shown). ATP molecule is hydrolyzed per 5 min per DnaK monomer) and isolating complexes of DnaK and nucleotide. We found DISCUSSION that DnaJ alone accelerated hydrolysis of DnaK-bound ATP, It whereas GrpE alone accelerated the release of ATP or ADP has been previously reported that DnaK interacts with a bound to DnaK. The net result ofadding both DnaJ and GrpE number of different proteins: AP, GrpE, and RNA polymer- was an acceleration in both the hydrolysis and the release of the DnaK-bound ATP. The findings that DnaJ and GrpE affect b 2 3 4 5 6789101112 A different aspects of DnaK-nucleotide interactions and that DnaJ and GrpE can exert their effects in the absence of each

ADP- # # . - other suggests that they may work sequentially: DnaJ binds to DnaK and accelerates the rate of ATP hydrolysis, then GrpE binds and accelerates the rate of release of the bound ADP. B Hence, there is no need to hypothesize the existence of an intracellular DnaK-DnaJ-GrpE complex. It could be that the stimulation of DnaK's ATPase activity is the only role that DnaJ and GrpE play in assisting DnaK function; alternatively, DnaJ and GrpE alone or together could also participate ATP- I directly in either the binding or the release of some polypep- c tides from DnaK. Nevertheless, our results reported here clearly explain why mutations in dnaK, dnaJ, or grpE often ADP- i em exhibit similar phenotypes (1, 2). The grpE280 and dnaK756 mutations were originally dis- ATP-. covered because they resulted in a block to bacteriophage A DNA replication at all temperatures (19, 28). The purified D DnaK756 protein was subsequently shown to be inactive in ADP- both crude and purified in vitro Adv DNA replication systems (17, 20). The inability ofDnaK756 mutant protein to function in the A replication system could be partly due to its inability ATP- to form a stable complex with either the AP replication protein (29) or the wild-type GrpE protein. The finding reported here, namely that DnaK756 protein exhibits 50-fold higher ATPase FIG. 5. Influence of DnaJ and GrpE on DnaK'nucleotide com- activity levels than wild type, is consistent with its inability plex. A solution of 2.2 AM DnaK with bound nucleotide was to function in Adv DNA replication-i.e., it is possible that incubated for 1 min at 300C as follows: A, alone; B, with DnaJ (3.1 DnaK756 can form an initial complex with the AP protein, but ,uM); C, with GrpE (2.4 ,uM); and D, with DnaJ (3.1 AtM) and GrpE such a complex dissociates rapidly (because of DnaK756's (2.4 AuM). Incubated mixtures were then filtered on a P-60 column. intrinsic ATPase to an unstable inter- The fractions (1-12) were collected and analyzed by thin-layer high activity), leading chromatography on PEI-cellulose, followed by autoradiography. action between the two proteins. Alternatively, the dnaK756 Controls: lane a, ADP/ATP distribution in the DnaK-nucleotide mutation may affect the intrinsic ability of the DnaK756 complex before the 1-min incubation; lane b, ADP/ATP distribution protein to recognize AP or other polypeptides as substrates. following the 1-min incubation. For more details, see text. We have recently shown that DnaK, with the help of hydro- Downloaded by guest on September 28, 2021 2878 Biochemistry: Liberek et al. Proc. Natl. Acad. Sci. USA 88 (1991) lyzable ATP, can act like a "molecular crowbar," disaggre- Stress Proteins in Biology and Medicine, eds. Morimoto, R., gating and restoring activity to an already aggregated form of Tissieres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., E. coli RNA polymerase holoenzyme (5). The mutant Cold Spring Harbor, NY), pp. 1-36. DnaK756 protein has lost the ability to "resurrect" such 4. Pelham, H. (1986) Cell 46, 959-961. RNA polymerase aggregates. Again, this may be due to either 5. Skowyra, D., Georgopoulos, C. & Zylicz, M. (1990) Cell 62, the inability to recognize and bind to the aggregated protein 939-944. 6. Zylicz, M., LeBowitz, J. H., McMacken, R. & Georgopoulos, or to "nonproductive" interactions resulting from the mutant C. (1983) Proc. Natl. Acad. Sci. USA 80, 6431-6435. protein's high intrinsic ATPase activity. Alternatively, both 7. Welch, W. J. & Feramisco, J. R. (1985) Mol. Cell. Biol. 5, processes could be affected. 1229-1237. The inability of GrpE280 protein to stimulate DnaK's 8. Lewis, M. J. & Pelham, H. (1985) EMBO J. 4, 3137-3143. ATPase activity or release the DnaK-bound ATP/ADP moi- 9. Hinds, P., Finlay, C., Frey, A. & Levine, A. J. (1987) Mol. ety is also interesting. It has been shown previously that the Cell. Biol. 7, 2863-2869. DnaK and GrpE280 proteins do not form an isolatable 10. Munro, S. & Pelham, H. R. B. (1986) Cell 46, 291-300. complex, as judged by coimmunoprecipitation (30) or cosed- 11. Chappell, T. G., Welch, W. J., Schlossman, D. M., Palter, imentation in a glycerol gradient (23). Nevertheless, DnaK K. G., Schlesinger, M. J. & Rothman, J. E. (1986) Cell 45, and GrpE280 do interact, as evidenced by the formation of 3-13. crosslinked complexes in the presence of glutaraldehyde. 12. Ungewickell, E. (1985) EMBO J. 4, 3385-3391. However, the DnaK-GrpE280 complex is highly unstable, 13. Beckmann, R. P., Mizzen, L. A. & Welch, W. J. (1990) Sci- ence 248, 850-854. dissociating rapidly if diluted prior to crosslinking (23). The 14. Flynn, G. C., Chappell, T. G. & Rothman, J. E. (1989) Science inability to form a stable complex could be the reason for 245, 385-390. GrpE280's failure to stimulate DnaK's ATPase activity. The 15. Alfano, C. & McMacken, R. (1989) J. Biol. Chem. 264, 10709- DnaK-GrpE protein interaction must be important to E. 10718. cOli's survival. We have previously shown that (i) extragenic 16. Zylicz, M., Ang, D., Liberek, K. & Georgopoulos, C. (1989) suppressors that allow grpE280 mutant bacteria to grow at EMBO J. 8, 1601-1608. 420C map predominantly in the dnaK gene (30), and (ii) the 17. Liberek, K., Georgopoulos, C. & Zylicz, M. (1988) Proc. Nati. grpE gene cannot be deleted in wild-type E. coli. However, Acad. Sci. USA 85, 6632-6636. the grpE gene can be deleted in certain E. coli bacteria that 18. Ang, D., Chandrasekhar, G. N., Zylicz, M. & Georgopoulos, carry compensatory mutations for dnaK defects, in as-yet- C. (1986) J. Bacteriol. 167, 25-29. unidentified genes (31). Hence, whatever mutations compen- 19. Georgopoulos, C. (1977) Mol. Gen. Genet. 151, 35-39. sate for the lack of DnaK function allow E. coli to simulta- 20. Zylicz, M. & Georgopoulos, C. (1984) J. Biol. Chem. 259, 8820-8825. neously lose the requirement for GrpE function. This result 21. Zylicz, M., Ang, D. & Georgopoulos, C. (1987) J. Biol. Chem. suggests that perhaps the only role of GrpE in E. coli is to 262, 17437-17442. modulate the various activities exhibited by DnaK. 22. Zylicz, M., Yamamoto, T., McKittrick, N., Sell, S. & Georg- opoulos, C. (1985) J. Biol. Chem. 260, 7591-7598. We thank Jeni Urry for expert and cheerful editing of the manu- 23. Ang, D. (1988) Ph.D. thesis (Univ. of Utah, Salt Lake City). script. K.L. thanks Dr. M. Kohiyama for his hospitality and ex- 24. Shlomai, J. & Kornberg, A. (1980) J. Biol. Chem. 255, 6789- change ofideas during K.L.'s brief stay in his laboratory in 1989. This 6793. work was supported by National Institutes of Health grants to C.G., 25. Bochner, B. R., Zylicz, M. & Georgopoulos, C. (1986) J. a National Institutes of Health training grant to D.A., and Polish Bacteriol. 168, 931-935. Ministry of Education Grant DB-0193-70/90 to M.Z. 26. Bardwell, J. C. A., Tilly, K., Craig, E., King, J., Zylicz, M. & Georgopoulos, C. (1986) J. Biol. Chem. 261, 1782-1785. 1. Georgopoulos, C., Ang, D., Liberek, K. & Zylicz, M. (1990) in 27. Clarke, C. F., Cheng, K., Frey, A. B., Stein, R., Hinds, P. W. Stress Proteins in Biology and Medicine, eds. Morimoto, R., & Levine, A. J. (1988) Mol. Cell. Biol. 8, 1206-1215. Tissieres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., 28. Saito, H. & Uchida, H. (1977) J. Mol. Biol. 113, 1-25. Cold Spring Harbor, NY), pp. 191-221. 29. Liberek, K., Osipiuk, J., Zylicz, M., Ang, D., Skorko, J. & 2. Gross, C. A., Straus, D. B., Erickson, J. W. & Yura, T. (1990) Georgopoulos, C. (1990) J. Biol. Chem. 265, 3022-3029. in Stress Proteins in Biology and Medicine, eds. Morimoto, R., 30. Johnson, C., Chandrasekhar, G. N. & Georgopoulos, C. (1989) Tissieres, A. & Georgopoulos, C. (Cold Spring Harbor Lab., J. Bacteriol. 171, 1590-1596. Cold Spring Harbor, NY), pp. 167-189. 31. Ang, D. & Georgopoulos, C. (1989) J. Bacteriol. 171, 2748- 3. Morimoto, R., Tissieres, A. & Georgopoulos, C. (1990) in 2755. Downloaded by guest on September 28, 2021