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Autoregulation of the Escherichia Coli Heat Shock Response By Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11019-11023, December 1993 Biochemistry Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins (negative autoregulation/protein-protein interactions) KRZYSZTOF LIBEREK*tt AND COSTA GEORGOPOULOS* *Departement de Biochimie Medicale, Centre Medical Universitaire, 1, rue Michel-Servet, 1211 Geneve 4, Switzerland; and tDivision of Biophysics, Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland Communicated by Werner Arber, August 23, 1993 (receivedfor review March 8, 1993) ABSTRACT All organisms respond to various forms of '"chaperone" (19), usually acts together with two other heat stress, including heat shock. The heat shock response has been shock proteins, DnaJ and GrpE, to constitute a "chaperone universally conserved from bacteria to humans. In Escherichia machine" (20). colithe heat shock response is under the positive transcriptional The DnaJ protein is another example of a heat shock control of the o32 polypeptide and involves transient acceler- protein that has been extensively conserved in evolution (21). ation in the rate of synthesis of a few dozen genes. Three of the Recently, it has been shown that E. coli DnaJ protein can heat shock genes-dnaK, dnaJ, and grpE-are special because suppress the import defect into the endoplasmic reticulum mutations in any one of these lead to constitutive levels of heat and mitochondria of YDJ1-defective yeast mutants (22), shock gene expression, implying that their products negatively demonstrating a functional conservation throughout evolu- autoregulate their own synthesis. The DnaK, DnaJ, and GrpE tion. DnaJ and GrpE work synergistically with DnaK in the proteins have been known to function in various biological replication of bacteriophages A and P1 (23-25). Part of the situations, including bacteriophage A replication. Here, we role of DnaJ protein is to target both the phage A P and RepA report the formation of an ATP hydrolysis-dependent complex replication proteins for DnaK action (26-28). In the case of of DnaJ, g32, and DnaK proteins in vitro. This DnaJ-og32- P1 replication the formation of the DnaJ-RepA protein com- DnaK complex has been seen under different conditions, plex is necessary for the subsequent action of DnaK, result- including glycerol gradient sedimentation and co-immunopre- ing in the monomerization ofRepA protein dimers (26). DnaJ cipitation. The DnaK and DnaJ proteins in the presence ofATP and GrpE also interact with DnaK directly. The GrpE protein can interfere with the efficient binding of a32 to the RNA forms a stable ATP-sensitive complex with DnaK, whereas polymerase core, and are capable of disrupting a preexisting GrpE and DnaJ together greatly stimulate the ATPase activ- g32-RNA polymerase complex. Our results suggest a possible ity of DnaK (29). mechanism for the autoregulation of the heat shock response. We have previously shown that purified DnaK but not DnaK756 mutant protein interacts with o.32 and that this All organisms respond to environmental stresses, such as interaction is sensitive to ATP hydrolysis (11). Recently, heat and ethanol, by the rapid and transient acceleration in Gamer et al. (14) showed, using crude extracts and affinity the rate of synthesis of a group ofproteins collectively called chromatography, that DnaK, DnaJ, and GrpE proteins also the heat shock proteins (for reviews see refs. 1-4). Esche- interact with or32 transcription factor. In this work we show richia coli possesses approximately 20 known heat shock that purified DnaK and DnaJ proteins form a stable complex genes whose expression is positively regulated by the oa32 with o.32 in the presence ofATP. Such an association prevents polypeptide (the rpoH gene product) at the transcriptional v32 from binding to RNA polymerase. In addition, DnaK and level. The o32 polypeptide is extremely unstable in vivo, with DnaJ can also actively "strip" vo32 from its complex with a half-life of 1 min at normal temperatures, its intracellular RNA polymerase. levels usually correlating with the magnitude of the heat shock response (refs. 5-7, reviewed in refs. 2 and 8). The heat MATERIALS AND METHODS shock respor .e is negatively autoregulated, since mutations in the dnaK, dnaJ, or grpE heat shock genes lead to an Protein Purification. DnaK, DnaJ, GrpE, a32, and RNA overproduction of the heat shock proteins. Such an overex- polymerase were purified according to refs. 11, 30, and 31. pression of the heat shock response is due in part to a The purity of all enzymes was >90% except o70 (-75%). stabilization of o:32 polypeptide in these genetic backgrounds Protein concentrations were estimated by the Bio-Rad assay (6, 7, 9). and the molar ratio was determined on the basis that all The major heat shock protein DnaK has been extremely proteins are monomers except DnaJ (dimer). conserved during evolution. It is 50% identical at the amino Glycerol Gradient Centrifugation. Reaction mixtures (60 acid sequence level to the eukaryotic Hsp7O protein. DnaK ,l) in buffer A (40 mM Hepes/KOH, pH 7.6/100 mM NaCl/1 can bind to various unfolded polypeptides, such as denatur- mM dithiothreitol/5 mM MgCl2/0.1 mM EDTA) were as- ated bovine pancreatic trypsin inhibitor, as well as seemingly sembled on ice. The mixtures were incubated for 10 min at folded polypeptides such as E. coli GrpE, a32, phage A P, and 37°C and then loaded directly onto a 3.2-ml linear 15-35% the human antitumor p53 protein (10-16). Such binding is (vol/vol) glycerol gradient in buffer A and centrifuged at disrupted in the presence of ATP. In addition, overproduc- 48,000 rpm for 24 hr at 2°C in a Beckman SW60 rotor. tion of DnaK aids the translocation of an export-deficient Fractions were collected from the bottom of the tube and MalB-LacZ hybrid protein (17), and DnaK/DnaJ overpro- analyzed by SDS/PAGE, followed by Coomassie blue stain- duction can substitute for some of the functions for the ing, silver staining, and/or Western blot analysis with anti- missing SecB protein (18). DnaK, also referred to as a bodies to o,32 and 125I-labeled staphylococcal protein A or a chemiluminescent detection system using Lumigen PPD as a substrate (Boehringer Mannheim). In the experiments with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be sent at the * address. 11019 Downloaded by guest on September 28, 2021 11020 Biochemistry: Liberek and Georgopoulos Proc. Natl. Acad Sci. USA 90 (1993) RNA polymerase, the glycerol gradient was increased to protein copurifies with or32 during Q-Sepharose chromatog- 20-40%o (vol/vol) and the centrifugation time was lowered to raphy (results not shown). Our results with purified proteins 20 hr. Where indicated, ATP was used at 1 mM in the reaction confirm and extend those of Gamer et al. (14), who demon- mixtures and at 100 ,uM throughout the gradient. strated a a32-DnaJ interaction by using histidine-tagged a32 Coimmunoprecipitation Experiments. The reaction mix- bound to a nickel affinity column and passing crude extracts tures in buffer A were assembled on ice and then incubated over it. for 15 min at room temperature. Ten microliters ofpolyclonal Formation of the DnaJ-cr32-DnaK Complex Is ATP Depen- anti-DnaK rabbit serum was added and the mixtures were dent. Next, we determined the relative affinity of o-32 for incubated for 1 hr on ice. Forty microliters of a 1:1 (vol/vol) DnaK or DnaJ. In the absence of ATP, when the molar ratio mixture of protein A-agarose in buffer B (10 mM Tris HCl, of DnaK, DnaJ, and or32 was approximately 1:1:1 in the pH 7.2/150 mM NaCl/5 mM EDTA/1% Triton X-100) was incubation mixture, the majority of or32 was found in a added. After an additional 30-min incubation on ice the complex with DnaJ only (Fig. 2). This result demonstrates protein A beads were washed five times with 500 ,ul of buffer that DnaJ binds to ao32 much better than DnaK does. This B. The or32 protein that associated with protein A-agarose was conclusion is consistent with the observation that, when the detected by SDS/PAGE, followed by Western blot analysis amount of DnaK in the preincubation mixture was increased with polyclonal antibodies to o-32 as described above. to 10-fold above that of o.32 and DnaJ, the formation of the DnaJ-or32-DnaK complex was readily seen. However, as expected, the bulk of DnaK sedimented in a position char- RESULTS acteristic of that of a DnaK monomer (results not shown). The g32 Transcription Factor Interacts with DnaJ Protein. A surprising observation made during the course of this Because we had previously demonstrated that the DnaK work was that the presence of ATP greatly stimulated the chaperone protein can form a weak, ATP hydrolysis- quantitative formation of DnaJ-cr32-DnaK complex even sensitive complex with oJ32 (11), we investigated whether the when the molar ratio of the three proteins was 1:1:1 (Fig. 2). DnaJ chaperone can also interact with cr32. Such a possible For the efficient formation of this complex, the presence of interaction between the DnaJ and ar32 protein was investi- ATP is absolutely required; i.e., when ATP was replaced gated by glycerol gradient centrifugation. Under such con- either by its nonhydrolyzable analogs adenosine 5'-l,3, ditions, DnaJ is known to sediment as a dimer (30), while cr32 imido]triphosphate and adenosine 5'-[y-thioltriphosphate or When was by ADP, the DnaJ-or32-DnaK complex did not form.
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