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Dnak, Dnaj^ and Grpe Heat Shock Proteins Negatively Regulate Heat Shock Gene Expression by Controlling the Synthesis and Stability of A^^

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Dnak, Dnaj^ and Grpe Heat Shock Proteins Negatively Regulate Heat Shock Gene Expression by Controlling the Synthesis and Stability of A^^ Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press DnaK, DnaJ^ and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of a^^ David Straus,* William Walter, and Carol A. Gross Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 USA The Escherichia coli DnaK heat shock protein has been identified previously as a negative regulator of E. coli heat shock gene expression. We report that two other heat shock proteins, DnaJ and GrpE, are also involved in the negative regulation of heat shock gene expression. Strains carrying defective dnaK, dnaj, or grpE alleles have enhanced synthesis of heat shock proteins at low temperature and fail to shut off the heat shock response after shift to high temperature. These regulatory defects are due to the loss of normal control over the synthesis and stability of CT^^, the alternate RNA polymerase tr-factor required for heat shock gene expression. We conclude that DnaK, DnaJ, and GrpE regulate the concentration of o-^^. We suggest that the synthesis of heat shock proteins is controlled by a homeostatic mechanism linking the function of heat shock proteins to the concentration of a^^. [Key Words: Heat shock proteins; DnaK heat shock gene expression; o^^] Received August 9, 1990; revised version accepted September 12, 1990. The induction of heat shock proteins following an and after temperature upshift, depends on the function abrupt increase in growth temperature has been ob­ of the rpoH(htpR) gene (Neidhardt and VanBogelen served in every cell type examined, including examples 1981; Yamamori and Yura 1982; Zhou et al. 1988). The from eubacterial, archaebacterial, and eukaryotic or­ product of this gene has been identified as a a-factor, o^^, ganisms (Schlesinger et al. 1982; Craig 1985). Heat which enables RNA polymerase to recognize the pro­ shock proteins are also induced by a variety of other moters for heat shock genes (Grossman et al. 1984; stresses, including exposure to ethanol, UV irradiation, Cowing et al. 1985; Fujita et al. 1987). In addition to di­ oxidative agents, viral infection, and the presence of ab­ recting RNA polymerase to the heat shock genes, o^^ normal proteins (for review, see Neidhardt and VanBo- also regulates their expression. A rapid and transient in­ gelen 1987). Recent work suggests that heat shock pro­ crease in the level of cr^^ is observed following tempera­ teins provide functions that are essential in the control ture upshift, which can account for the induction of heat of protein folding (Chirico et al. 1988; Deshaies et al. shock proteins (Lesley et al. 1987; Skelly et al. 1987; 1988; Goloubinoff et al. 1989a; Gaitanaris et al. 1990), Straus et al. 1987). Additionally, artificial induction of assembly (Goloubinoff et al. 1989b), and disassembly (7^2 without a temperature shift results in the induction (Alfano and McMacken 1989; Dodson et al. 1989; Zylicz of heat shock proteins (Grossman et al. 1987). The in­ et al. 1989). It is likely that heat shock proteins carry out crease in (T^^ level that accompanies a temperature up­ similar functions in most cells, as there is a high degree shift occurs as a result of changes in both the stability of sequence homology between heat shock proteins from and synthesis of o^^ (Straus et al. 1987). It is not known widely divergent organisms (Bardwell and Craig 1984, how an abrupt change in temperature causes an increase 1987; McMullin and Hallberg 1988). in the synthesis and stability of the alternate a-factor. When Escherichia coli are shifted from low to high Tilly et al. (1983) observed that a mutation in the growth temperature, —17 heat shock proteins are in­ dnaK heat shock gene resulted in enhanced heat shock duced (Neidhardt and VanBogelen 1987). This induction gene expression at low temperature and an extended is transient, peaking at 5-15 min after upshift and the heat shock response after shift to high temperature, sug­ dropping to a new steady-state rate of synthesis, which gesting that DnaK is a negative regulator of the heat is characteristic of the new growth temperature. The ex­ shock response. We have examined the phenotype of pression of heat shock proteins, both at low temperature other heat shock gene mutants besides dnaK and found that dnaf and grpE mutants have a similar effect on heat ^Present address: Howard Hughes Medical Institute, University of Cali­ shock gene expression. We have determined the mecha­ fornia School of Medicine, San Francisco, California 94143 USA. nism for this effect; all three mutants are defective in 2202 GENES & DEVELOPMENT 4:2202-2209 © 1990 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/90 $1.00 Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press DnaK, DnaJ, and GrpE regulate concentration of <T*^ the control of o^^ synthesis and stability. These results 14 r indicate that heat shock gene expression is controlled by a negative feedback loop in which heat shock proteins regulate their own synthesis by controlling the level of CT^^. We suggest that temperature changes are sensed by this homeostatic regulatory mechanism through alter­ ation in the function of DnaK, DnaJ, and GrpE. Results Expression of heat shock genes is enhanced in dnaj and grpE mutants The regulation of heat shock protein synthesis is altered Min at 42° in two ways in strains carrying the dnaK756 allele (Tilly Figure 1. Shutoff of the heat shock response is defective in the et al. 1983). First, synthesis of heat shock proteins in dnaK756, grpE280, and dna]259 mutants. The relative syn­ these strains is increased during growth at 30°C. Second, thesis of GroEL in wild-type (•), dnaK756 (O), grpE280 (•), and the shutoff phase of the heat shock response is delayed. dna]259 (D) strains was determined at 30°C or at 10, 20, and 60 To determine whether this effect is specific to the dnaK min after shift to 42°C by pulse-labeling and two-dimensional mutant, we examined the synthesis of heat shock pro­ gel electrophoresis, as described in Materials and methods. The teins in strains carrying mutations in other heat shock synthesis rates are normalized to the 30°C wild-type rate. genes. Our results indicate that of the strains analyzed, Shutoff of DnaK, HtpG, and HtpM synthesis was also defective only mutations in dnaJ or gipE have a significant effect in the mutants, and the relative magnitude of the defects in the on heat shock protein synthesis. different mutants was the same as that observed for GroEL syn­ Heat shock protein synthesis in the mutant strains thesis. was analyzed by two-dimensional gel electrophoresis of extracts from cells that had been pulse-labeled with ra­ dioactive amino acids at 30°C or at various times after and gipE heat shock genes also result in altered syn­ shift to 42°C. As compared to the parental strain, the thesis of heat shock proteins at both 30°C and during dnaK756, dnaf259, and grpE280 strains all showed a shutoff of the heat shock response. two- to fourfold increase in the 30°C synthesis rate of Synthesis of heat shock proteins in strains carrying the four heat shock proteins analyzed. This effect was mutations in other heat shock genes was also examined. specific to the heat shock proteins, as the synthesis of The lonl46 :: TnlO, gioELUO, and gioESSO alleles had EF-Tu (Table 1) and other non-heat shock proteins (data only small effects on heat shock protein synthesis. not shown) was unaltered in the mutants. As is the case These strains showed an increase of —50% in heat shock for dnaK756, the dna]259 and gipE280 mutants were protein synthesis at 30°C and exhibited only a slight also defective in the shutoff phase of the heat shock re­ delay in shutting off the heat shock response. Data for sponse following shift from 30 to 42°C. As exemplified the lonl46 :: TnlO strain is shown in Figure 2. by the induction kinetics of the GroEL heat shock pro­ The induction of heat shock protein synthesis fol­ tein, each of the mutants show only a gradual drop in lowing temperature upshift is the result of increased heat shock protein synthesis (Fig. 1). GroEL synthesis heat shock gene transcription (Yamamori and Yura remains elevated for at least 60 min after temperature 1982; Taylor et al. 1984; Cowing et al. 1985). To deter­ shift in the mutants. In wild-type cells, induction of mine whether altered heat shock gene expression in the GroEL is transient, peaking at ~5 min after temperature mutants could be explained by altered rates of transcrip­ upshift. The shutoff of synthesis of the other heat shock tion, we examined derivatives of dnaK756, dnaJ259, and proteins followed similar kinetics in the mutant strains gipE280, which carried a plasmid in which the expres­ (data not shown). These findings indicate that dnaK is sion of galactokinase is driven by the gioE promoter. In not the only heat shock gene involved in the regulation these strains the rate of galactokinase synthesis indi­ of heat shock protein synthesis. Mutations in the dnaJ cates the rate of transcription initiation at the groE pro- Table 1. Heat shock protein synthesis is enhanced in heat shock gene mutants at 30°C DnaK GroEL HtpG HtpM EF-Tu Wild type 1 ± 0.03 1 ± 0.05 1 ± 0.01 1 ± 0.11 1 ± 0.06 dnaK756 L93 ± 0.04 1.93 ± 0.02 2.28 ± 0.15 1.59 ± 0.12 0.95 ± 0.01 gipE280 2.54 ± 0.20 2.07 ± 0.03 2.46 ± 0.06 1.82 ± 0.20 0.98 ± 0.01 dnaJ259 4.40 ± 0.33 2.59 ± 0.06 4.03 ± 0.20 2.90 ± 0.41 1.00 ± 0.01 The relative synthesis rate of the DnaK, GroEL, HtpG, and HtpM heat shock proteins and EF-Tu were determined in isogenic deriva­ tives of C600, as described in Materials and methods.
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