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. 1997 Dec;179(23):7219–7225. doi: 10.1128/jb.179.23.7219-7225.1997

Synergistic roles of HslVU and other ATP-dependent proteases in controlling in vivo turnover of sigma32 and abnormal proteins in Escherichia coli.

M Kanemori 1, K Nishihara 1, H Yanagi 1, T Yura 1
PMCID: PMC179669  PMID: 9393683

Abstract

Production of abnormal proteins during steady-state growth induces the heat shock response by stabilizing normally unstable sigma32 (encoded by the rpoH gene) specifically required for transcription of heat shock genes. We report here that a multicopy plasmid carrying the hslVU operon encoding a novel ATP-dependent protease inhibits the heat shock response induced by production of human prourokinase (proUK) in Escherichia coli. The overproduction of HslVU (ClpQY) protease markedly reduced the stability and accumulation of proUK and thus reduced the induction of heat shock proteins. In agreement with this finding, deletion of the chromosomal hslVU genes significantly enhanced levels of proUK and sigma32 without appreciably affecting cell growth. When the deltahslVU deletion was combined with another protease mutation (lon, clpP, or ftsH/hflB), the resulting multiple mutations caused higher stabilization of proUK and sigma32, enhanced synthesis of heat shock proteins, and temperature-sensitive growth. Furthermore, overproduction of HslVU protease reduced sigma32 levels in strains that were otherwise expected to produce enhanced levels of sigma32 due either to the absence of Lon-ClpXP proteases or to the limiting levels of FtsH protease. Thus, a set of ATP-dependent proteases appear to play synergistic roles in the negative control of the heat shock response by modulating in vivo turnover of sigma32 as well as through degradation of abnormal proteins.

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Selected References

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  1. Akiyama Y., Ogura T., Ito K. Involvement of FtsH in protein assembly into and through the membrane. I. Mutations that reduce retention efficiency of a cytoplasmic reporter. J Biol Chem. 1994 Feb 18;269(7):5218–5224. [PubMed] [Google Scholar]
  2. Bukau B. Regulation of the Escherichia coli heat-shock response. Mol Microbiol. 1993 Aug;9(4):671–680. doi: 10.1111/j.1365-2958.1993.tb01727.x. [DOI] [PubMed] [Google Scholar]
  3. Chuang S. E., Burland V., Plunkett G., 3rd, Daniels D. L., Blattner F. R. Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli. Gene. 1993 Nov 30;134(1):1–6. doi: 10.1016/0378-1119(93)90167-2. [DOI] [PubMed] [Google Scholar]
  4. Craig E. A., Gross C. A. Is hsp70 the cellular thermometer? Trends Biochem Sci. 1991 Apr;16(4):135–140. doi: 10.1016/0968-0004(91)90055-z. [DOI] [PubMed] [Google Scholar]
  5. Gamer J., Bujard H., Bukau B. Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32. Cell. 1992 May 29;69(5):833–842. doi: 10.1016/0092-8674(92)90294-m. [DOI] [PubMed] [Google Scholar]
  6. Goff S. A., Goldberg A. L. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell. 1985 Jun;41(2):587–595. doi: 10.1016/s0092-8674(85)80031-3. [DOI] [PubMed] [Google Scholar]
  7. Goldberg A. L. The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur J Biochem. 1992 Jan 15;203(1-2):9–23. doi: 10.1111/j.1432-1033.1992.tb19822.x. [DOI] [PubMed] [Google Scholar]
  8. Gottesman S., Maurizi M. R. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev. 1992 Dec;56(4):592–621. doi: 10.1128/mr.56.4.592-621.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gottesman S. Proteases and their targets in Escherichia coli. Annu Rev Genet. 1996;30:465–506. doi: 10.1146/annurev.genet.30.1.465. [DOI] [PubMed] [Google Scholar]
  10. Hartl F. U. Molecular chaperones in cellular protein folding. Nature. 1996 Jun 13;381(6583):571–579. doi: 10.1038/381571a0. [DOI] [PubMed] [Google Scholar]
  11. Herman C., Thévenet D., D'Ari R., Bouloc P. Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3516–3520. doi: 10.1073/pnas.92.8.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ito K., Akiyama Y., Yura T., Shiba K. Diverse effects of the MalE-LacZ hybrid protein on Escherichia coli cell physiology. J Bacteriol. 1986 Jul;167(1):201–204. doi: 10.1128/jb.167.1.201-204.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kanemori M., Mori H., Yura T. Induction of heat shock proteins by abnormal proteins results from stabilization and not increased synthesis of sigma 32 in Escherichia coli. J Bacteriol. 1994 Sep;176(18):5648–5653. doi: 10.1128/jb.176.18.5648-5653.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kessel M., Wu W., Gottesman S., Kocsis E., Steven A. C., Maurizi M. R. Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY. FEBS Lett. 1996 Dec 2;398(2-3):274–278. doi: 10.1016/s0014-5793(96)01261-6. [DOI] [PubMed] [Google Scholar]
  15. Liberek K., Galitski T. P., Zylicz M., Georgopoulos C. The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3516–3520. doi: 10.1073/pnas.89.8.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lupas A., Zwickl P., Baumeister W. Proteasome sequences in eubacteria. Trends Biochem Sci. 1994 Dec;19(12):533–534. doi: 10.1016/0968-0004(94)90054-x. [DOI] [PubMed] [Google Scholar]
  17. Maurizi M. R. Proteases and protein degradation in Escherichia coli. Experientia. 1992 Feb 15;48(2):178–201. doi: 10.1007/BF01923511. [DOI] [PubMed] [Google Scholar]
  18. Missiakas D., Schwager F., Betton J. M., Georgopoulos C., Raina S. Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli. EMBO J. 1996 Dec 16;15(24):6899–6909. [PMC free article] [PubMed] [Google Scholar]
  19. Nagai H., Yuzawa H., Yura T. Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10515–10519. doi: 10.1073/pnas.88.23.10515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Parsell D. A., Sauer R. T. Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation. Genes Dev. 1989 Aug;3(8):1226–1232. doi: 10.1101/gad.3.8.1226. [DOI] [PubMed] [Google Scholar]
  21. Qu J. N., Makino S. I., Adachi H., Koyama Y., Akiyama Y., Ito K., Tomoyasu T., Ogura T., Matsuzawa H. The tolZ gene of Escherichia coli is identified as the ftsH gene. J Bacteriol. 1996 Jun;178(12):3457–3461. doi: 10.1128/jb.178.12.3457-3461.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rohrwild M., Coux O., Huang H. C., Moerschell R. P., Yoo S. J., Seol J. H., Chung C. H., Goldberg A. L. HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5808–5813. doi: 10.1073/pnas.93.12.5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shirai Y., Akiyama Y., Ito K. Suppression of ftsH mutant phenotypes by overproduction of molecular chaperones. J Bacteriol. 1996 Feb;178(4):1141–1145. doi: 10.1128/jb.178.4.1141-1145.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stahl F. W., Kobayashi I., Thaler D., Stahl M. M. Direction of travel of RecBC recombinase through bacteriophage lambda DNA. Genetics. 1986 Jun;113(2):215–227. doi: 10.1093/genetics/113.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Straus D. B., Walter W. A., Gross C. A. The heat shock response of E. coli is regulated by changes in the concentration of sigma 32. Nature. 1987 Sep 24;329(6137):348–351. doi: 10.1038/329348a0. [DOI] [PubMed] [Google Scholar]
  26. Straus D., Walter W., Gross C. A. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev. 1990 Dec;4(12A):2202–2209. doi: 10.1101/gad.4.12a.2202. [DOI] [PubMed] [Google Scholar]
  27. Tilly K., McKittrick N., Zylicz M., Georgopoulos C. The dnaK protein modulates the heat-shock response of Escherichia coli. Cell. 1983 Sep;34(2):641–646. doi: 10.1016/0092-8674(83)90396-3. [DOI] [PubMed] [Google Scholar]
  28. Tobe T., Ito K., Yura T. Isolation and physical mapping of temperature-sensitive mutants defective in heat-shock induction of proteins in Escherichia coli. Mol Gen Genet. 1984;195(1-2):10–16. doi: 10.1007/BF00332716. [DOI] [PubMed] [Google Scholar]
  29. Tomoyasu T., Gamer J., Bukau B., Kanemori M., Mori H., Rutman A. J., Oppenheim A. B., Yura T., Yamanaka K., Niki H. Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32. EMBO J. 1995 Jun 1;14(11):2551–2560. doi: 10.1002/j.1460-2075.1995.tb07253.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wawrzynow A., Wojtkowiak D., Marszalek J., Banecki B., Jonsen M., Graves B., Georgopoulos C., Zylicz M. The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. EMBO J. 1995 May 1;14(9):1867–1877. doi: 10.1002/j.1460-2075.1995.tb07179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wild J., Walter W. A., Gross C. A., Altman E. Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response. J Bacteriol. 1993 Jul;175(13):3992–3997. doi: 10.1128/jb.175.13.3992-3997.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yano R., Imai M., Yura T. The use of operon fusions in studies of the heat-shock response: effects of altered sigma 32 on heat-shock promoter function in Escherichia coli. Mol Gen Genet. 1987 Apr;207(1):24–28. doi: 10.1007/BF00331486. [DOI] [PubMed] [Google Scholar]
  33. Yano R., Nagai H., Shiba K., Yura T. A mutation that enhances synthesis of sigma 32 and suppresses temperature-sensitive growth of the rpoH15 mutant of Escherichia coli. J Bacteriol. 1990 Apr;172(4):2124–2130. doi: 10.1128/jb.172.4.2124-2130.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yoo S. J., Seol J. H., Shin D. H., Rohrwild M., Kang M. S., Tanaka K., Goldberg A. L., Chung C. H. Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli. J Biol Chem. 1996 Jun 14;271(24):14035–14040. doi: 10.1074/jbc.271.24.14035. [DOI] [PubMed] [Google Scholar]
  35. Yura T., Nagai H., Mori H. Regulation of the heat-shock response in bacteria. Annu Rev Microbiol. 1993;47:321–350. doi: 10.1146/annurev.mi.47.100193.001541. [DOI] [PubMed] [Google Scholar]
  36. Yura T. Regulation and conservation of the heat-shock transcription factor sigma32. Genes Cells. 1996 Mar;1(3):277–284. doi: 10.1046/j.1365-2443.1996.28028.x. [DOI] [PubMed] [Google Scholar]
  37. Yuzawa H., Nagai H., Mori H., Yura T. Heat induction of sigma 32 synthesis mediated by mRNA secondary structure: a primary step of the heat shock response in Escherichia coli. Nucleic Acids Res. 1993 Nov 25;21(23):5449–5455. doi: 10.1093/nar/21.23.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]

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