The genes that encode the subunits of the Clp protease of Escherichia coli, cipA and clpP, appear to be regulated differently from each other. The cipA gene does not seem to be under heat shock control (Y. S. Katayama, S. Gottesman, J. Pumphrey, S. Rudikoff, W. P. Clark, and M. R. Maurizi, J. Biol. Chem. 263:15226-15236, 1988). In contrast, the level of ClpP protein was increased in rpoH+ cells but not in null rpoH cells after an upshift in temperature from 17 to 43°C. The level of ClpP protein in a null dnaK strain was also elevated relative to the level of ClpP protein in an otherwise isogenic dnaK+ strain. In two-dimensional gels, the ClpP protein was located in the position of the previously unidentified heat shock protein F21.5. No protein spot corresponding to F21.5 was present in two-dimensional gels of a null clpP strain. The clpP gene, therefore, appears to be a heat shock gene, expressed in a &32-dependent manner and negatively regulated by DnaK; the product of clpP is the previously unidentified heat shock protein F21.5. When Escherichia coli cultures are shifted to an elevated growth temperature, a set of at least 17 heat shock proteins appears to be synthesized at an increased rate (26). The increased synthesis of these heat shock proteins depends upon the rpoH-encoded RNA polymerase cofactor au32, which specifically directs transcription from heat shock promoters (13). u32 is a positive effector of the heat shock response. DnaK protein, on the other hand, is a negative regulator of the E. coli heat shock response (41). In wild-type cells, the increased synthesis of heat shock proteins that occurs very soon after a temperature upshift gradually decreases to a new steady-state level by about 10 min after the upshift (48). dnaK mutant strains, however, do not turn off the heat shock response after a temperature upshift; dnaK mutants synthesize high levels of heat shock proteins at all temperatures (41).Neidhardt et al. (26) have located many heat shock proteins in autoradiographs of two-dimensional gels of extracts of heat-shocked E. coli cells. Through genetic and biochemical techniques, the identities of at least eight of the heat shock proteins found by autoradiography have been determined: GrpE (1), GroES (42), GroEL (24), DnaK (9), c70 (40), Lysyl-tRNA synthetase (14), DnaJ (4), and Lon (34).Lon, the well-characterized ATP-dependent protease of E. coli (8,12,23,49), is the only protease among the known heat shock proteins. A second ATP-dependent protease, referred to as Clp, has also been described (15,18). The Clp protease is composed of two components: an ATPase component, ClpA (16,17), and a proteolytic component, ClpP (16). Quantitation of immunoprecipitated ClpA protein from heat-shocked E. coli cells did not detect an increase in ClpA protein at elevated temperatures (17). Furthermore, ClpA synthesis remained unchanged at increased temperatures in a o2-defective strain, whereas the synthesis of heat shock proteins decreased (17). These observations led Katayama et al. (17) to conclude...
Extracts made from Escherichia coli null dnaK strains contained elevated levels of ATP-dependent proteolytic activity compared with levels in extracts made from dnaK+ strains. This ATP-dependent proteolytic activity was not due to Lon, Clp, or Alp-associated A third ATP-dependent protease, Clp, is composed of an ATPase component, ClpA, and a proteolytic component, ClpP (13,14). The function of the Clp protease is not known; in contrast to lon mutants, clpA mutants do not display any obvious phenotypes, and when assayed in vivo, they are not defective in the degradation of misfolded polypeptides (14).ClpP protein and Lon protease appear to be heat shock proteins (8,17,29). Cells exposed to an upshift in growth temperature show a rapid, but brief, increase in the synthesis of a small set of heat shock proteins, after which the level of synthesis decreases to a new steady-state level (26). Initiation of transcription of most heat shock genes is directed by r32, the product of the rpoH gene (10), in association with RNA polymerase. Recently another RNA polymerase cofactor, cE, has been described (6). crE initiates transcription at P3 heat shock promoters (6). Transcription of the U32 independent htrA gene is under aE control (6, 18), as is transcription of the rpoH gene at 50°C (6).Heat shock proteins in addition to Lon and Clp proteases seem to be involved in protein degradation (33 strains (16, 33). DnaK protein appears to regulate the heat shock response by interacting with the (u32 protein (11,36). Since the DnaK protein is a negative regulator of the heat shock response, dnaK mutants contain increased levels of heat shock proteins (35). Thus, DnaK-mediated effects on protein degradation may be secondary to changes in the levels of heat shock proteins.We have found that an ATP-dependent proteolytic activity in E. coli cell extracts, not due to the Lon, Clp, or Alp protease, is elevated in extracts of null dnaK cells. This increase in proteolytic activity is not C32 dependent. MATERIALS AND METHODSBacterial strains and media. The bacterial strains used in these experiments are described in
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