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 genes have been previously shown member of the hsp70 family) and p53 (an anti-oncogenic to be essential for bacteriophage 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 heat shock protein 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 gene 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 bacteria 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.