The EMBO Journal vol. 12 no. 1 1 pp.4137 - 4144, 1993 DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage Hartwig Schroder', Thomas Langer2, At least for the Escherichia coli homologs DnaK and GroEL, Franz-Ulrich Hartl2 and Bernd Bukau1l3 ATP-dependent substrate release appears to be controlled by the specific cofactors DnaJ and GrpE (for DnaK) and IZentrum fur Molekulare Biologie, Universitat Heidelberg, INF 282, GroES (for GroEL) (Goloubinoff et al., 1989; Liberek D69120 Heidelberg, Germany and 2Program in Cellular Biochemistry et al., 1991; Martin et al., 1991). In the case of DnaJ and and Biophysics, Rockefeller Research Laboratory, Sloan Kettering Institute, 1275 York Avenue, New York, NY 10021, USA GrpE, these cofactors act co-operatively to stimulate the ATPase activity of DnaK (Liberek et al., 1991). 3Corresponding author Interestingly, DnaJ has the capability to bind protein Communicated by H.Bujard substrates independently of DnaK, such as DnaB helicase (Georgopoulos et al., 1990; Liberek et al., 1990), heat Members of the conserved Hsp7O chaperone family are shock transcription factor a32 (Gamer et al., 1992), XP assumed to constitute a main cellular system for the (Georgopoulos et al., 1990), plasmid P1-encoded RepA prevention and the amelioration of stress-induced protein (Wickner, 1990), and unfolded rhodanese (Langer et al., damage, though little direct evidence exists for this 1992). The functional significance of this substrate binding function. We investigated the roles of the DnaK (Hsp7O), by DnaJ is unclear. DnaJ and GrpE chaperones of Escherichia coli in Given their capacity to assist protein folding, it is not prevention and repair of thermally induced protein surprising that Hsp7O chaperones have multiple roles in the damage using firefly luciferase as a test substrate. In vivo, metabolism of unstressed as well as of stressed cells (Bukau luciferase was rapidly inactivated at 42°C, but was and Walker, 1989a,b; Gething and Sambrook, 1992). One efficiently reactivated to 50% of its initial activity during particular role hypothesized for members of the Hsp7O subsequent incubation at 30°C. DnaK, DnaJ and GrpE family is that they prevent and/or repair stress-induced did not prevent luciferase inactivation, but were essential damage to proteins. While such a function is consistent with for its reactivation. In vitro, reactivation of heat- the concept of chaperones, little direct evidence exists for inactivated luciferase to 80% of its initial activity required it. The bulk of the information comes from studies of E. coli the combined activity of DnaK, DnaJ and GrpE as well DnaK, which has been shown to bind to various aberrant as ATP, but not GroEL and GroES. Dnaj associated with proteins including protein fragments (Straus et al., 1988; denatured luciferase, targeted DnaK to the substrate and Hellebust et al., 1990), mutant proteins (Clark et al., 1988; co-operated with DnaK to prevent luciferase aggregation Sherman and Goldberg, 1992) and unfolded proteins (Langer at 42°C, an activity that was required for subsequent et al., 1992). In addition, DnaK appears to be sufficient in reactivation. The protein repair function of DnaK, GrpE vitro to protect RNA polymerase from thermal inactivation and, in particular, DnaJ is likely to be part of the role and to reactivate thermally inactivated RNA polymerase, of these proteins in regulation of the heat shock response. though these activities require a large excess of DnaK Key words: Escherichia coli/GroEL/heat shock proteins/ (Skowyra et al., 1990). Finally, it has been demonstrated Hsp7O/protein folding that strains carrying mutations in dnaK, dnaJ or grpE are defective in the reactivation of a mutant form of X-repressor (Gaitanaris et al., 1990). We performed an analysis of the roles of major E.coli Introduction chaperones in prevention and repair of stress-induced protein Correct folding of proteins in cells often requires the damage. We demonstrate that DnaK, DnaJ and GrpE form assistance of molecular chaperones such as the Hsp7O a chaperone machinery that is essential for the efficient in (DnaK) and Hsp6O (GroEL) heat shock proteins (Gething vivo repair of a thermally denatured test protein, luciferase, and Sambrook, 1992; Hartl et al., 1992). Members of the and is sufficient for in vitro repair. DnaJ has a crucial Hsp7O family maintain proteins in unfolded states, cause the function in this process in that its binding to denatured dissociation of protein complexes and have been reported luciferase suppresses luciferase aggregation and targets DnaK to mediate the reactivation of an oligomeric enzyme (Gething to the substrate. Our data provide further support for a central and Sambrook, 1992; Skowyra et al., 1990). Members of role of DnaJ in directly transducing stress-induced protein the Hsp6O family promote the correct folding of proteins damage to induction of heat shock gene transcription. imported into cell organelles as well as the association of oligomeric proteins (Ellis and van der Vies, 1991; Zeilstra Results et al., 1991; Gething and Sambrook, 1992). Hsp7O and Hsp6O bind to fully or partially unfolded polypeptides and Role of heat shock proteins in thermal inactivation of maintain them in loosely folded conformations, thereby luciferase in vivo preventing non-productive folding reactions. The release of As a suitable thermolabile test substrate for investigation of substrates from these chaperones usually requires ATP the role of bacterial chaperones in prevention and repair of hydrolysis (Flynn et al., 1989; Goloubinoff et al., 1989). thermal denaturation of proteins both in vivo and in vitro Oxford University Press 4137 H.Schroder et al. we considered Photinus pyralis luciferase since it fulfils a number of relevant criteria: (i) it is a thermolabile, monomeric protein (Subramani and DeLuca, 1988; Nguyen et al., 1989); (ii) thermally inactivated luciferase produced in cultured mammalian cells can be reactivated in vivo (Pinto et al., 1991); (iii) its enzymatic activity, readily determined by a sensitive and highly reliable photometric assay (de Wet et al., 1987), can be utilized to monitor its folding status 40- even at low concentrations; (iv) activity measurements can be performed with whole E. coli cells expressing luciferase .e 20 (Knaus, 1991); and (v) the lack of a physiological role of luciferase in E. coli permits study of its thermal inactivation Cd 0 and reactivation in vivo without compromising essential metabolic functions. 80 We first determined the degree of thermolability of luciferase expressed in E. coli. For this purpose we used wild 4.0 type cells (MC4100) which carry pUHE multicopy plasmids 60 expressing the luciferase gene under control of the 20 Piac/lacO3 promoter/operator system (pUHEl 1-3). Due to deletion ofthe lacI gene in these cells, the plasmid-encoded 60 luciferase gene is constitutively expressed, although at a low level, which leads to accumulation of luciferase to low levels (data not shown). Cells grown at 30°C were treated with 0 2 4 6 8 10 high concentrations of kanamycin (100 Ag/ml), which is known to prevent completely any further protein biosynthesis time at 42C (mn) in E.coli (VanBogelen and Neidhardt, 1990), and were then transferred to heat shock temperatures followed by the Fig. 1. Thermal inactivation of luciferase in E.coli. (A) Wild type determination of luciferase activity at various times after the (MC4100; ]) and AdnaK52 mutant (BB1553; *) cells carrying luciferase-encoding pUHE11-3 were grown at 30°C to logarithmic temperature shift. The complete block of protein synthesis phase, then treated with kanamycin (100 /tg/ml) followed by incubation in the cells used in these experiments was verified by protein at 42°C. Luciferase activities were determined before and at the labeling experiments (data not shown). A heat shock to indicated times after shift to 42°C. (B) Wild type cells carrying the temperatures as low as 40°C was sufficient to inactivate luciferase gene in the chromosome (BB3030) and pUHE14-1 (grpE+) luciferase. The time course of was (A) or Bluescript (dnaK+dnaJ+) (0) plasmids or no plasmid (A) inactivation temperature were grown at 30°C to early logarithmic phase. Then, IPTG (1 mM) dependent, the half-time of inactivation being 5 min at 40°C, was added for 2 h followed by a shift of the cultures to 420C. 2 min at 42°C (see Figure 1A) and <1 min at 44°C. Thus, Luciferase activities of the cells were determined as for A. luciferase is rapidly inactivated at temperatures well within the growth temperature range of E. coli. Bluescript plasmids expressing the dnaKJ operon under We then investigated the possibility that heat shock proteins control of P,ac. The cellular concentrations of DnaK and provide a limited protection of luciferase from thermal DnaJ were raised by adding IPTG to the cultures at 30°C inactivation in wild type cells. We therefore tested whether which for DnaK led to an increase of at least 8-fold (for 1 the activity of luciferase during steady state growth at 30°C mM IPTG for 2 h) in its cellular level (>10% of total and the kinetics of its inactivation by heat are affected by cellular protein, as estimated from polyacrylamide gels of the AdnaK52, dnaJ259, grpE280, groEL100 and groES30 total cellular extracts) as compared with uninduced cells. null and missense mutations. These alleles either lead to loss Overproduction of DnaK alone (data not shown) or of DnaK of the protein (AdnaK52) or result in the production of gene and DnaJ did not provide any protection of luciferase from products that are functionally defective at high temperatures thermal inactivation at 420C (Figure 1B). We then and lead to temperature sensitive growth ofE. coli. In strains considered the possibility that GrpE is limiting for carrying these alleles only minor differences in luciferase DnaK/DnaJ function and that its overproduction might activity (< 3-fold) as compared with wild type were detected increase their efficiency.