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. 1992 Jan;11(1):71–77. doi: 10.1002/j.1460-2075.1992.tb05029.x

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

Sherman MYu 1, A L Goldberg 1
PMCID: PMC556427  PMID: 1740117

Abstract

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker T. A., Grossman A. D., Gross C. A. A gene regulating the heat shock response in Escherichia coli also affects proteolysis. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6779–6783. doi: 10.1073/pnas.81.21.6779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
  3. Cegielska A., Georgopoulos C. Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis. J Biol Chem. 1989 Dec 15;264(35):21122–21130. [PubMed] [Google Scholar]
  4. Chin D. T., Goff S. A., Webster T., Smith T., Goldberg A. L. Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. J Biol Chem. 1988 Aug 25;263(24):11718–11728. [PubMed] [Google Scholar]
  5. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  6. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  7. Goff S. A., Casson L. P., Goldberg A. L. Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6647–6651. doi: 10.1073/pnas.81.21.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goff S. A., Goldberg A. L. An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli. J Biol Chem. 1987 Apr 5;262(10):4508–4515. [PubMed] [Google Scholar]
  9. 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]
  10. Goldberg A. L., St John A. C. Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annu Rev Biochem. 1976;45:747–803. doi: 10.1146/annurev.bi.45.070176.003531. [DOI] [PubMed] [Google Scholar]
  11. Gottesman S. Genetics of proteolysis in Escherichia coli*. Annu Rev Genet. 1989;23:163–198. doi: 10.1146/annurev.ge.23.120189.001115. [DOI] [PubMed] [Google Scholar]
  12. Grossman A. D., Erickson J. W., Gross C. A. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. doi: 10.1016/0092-8674(84)90493-8. [DOI] [PubMed] [Google Scholar]
  13. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  14. Hurtley S. M., Bole D. G., Hoover-Litty H., Helenius A., Copeland C. S. Interactions of misfolded influenza virus hemagglutinin with binding protein (BiP). J Cell Biol. 1989 Jun;108(6):2117–2126. doi: 10.1083/jcb.108.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keller J. A., Simon L. D. Divergent effects of a dnaK mutation on abnormal protein degradation in Escherichia coli. Mol Microbiol. 1988 Jan;2(1):31–41. doi: 10.1111/j.1365-2958.1988.tb00004.x. [DOI] [PubMed] [Google Scholar]
  16. Kowit J. D., Goldberg A. L. Intermediate steps in the degradation of a specific abnormal protein in Escherichia coli. J Biol Chem. 1977 Dec 10;252(23):8350–8357. [PubMed] [Google Scholar]
  17. Kroh H. E., Simon L. D. The ClpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5. J Bacteriol. 1990 Oct;172(10):6026–6034. doi: 10.1128/jb.172.10.6026-6034.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Liberek K., Marszalek J., Ang D., Georgopoulos C., Zylicz M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2874–2878. doi: 10.1073/pnas.88.7.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lipinska B., Zylicz M., Georgopoulos C. The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase. J Bacteriol. 1990 Apr;172(4):1791–1797. doi: 10.1128/jb.172.4.1791-1797.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Menon A. S., Goldberg A. L. Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. J Biol Chem. 1987 Nov 5;262(31):14929–14934. [PubMed] [Google Scholar]
  22. Michaelis S., Hunt J. F., Beckwith J. Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli. J Bacteriol. 1986 Jul;167(1):160–167. doi: 10.1128/jb.167.1.160-167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Neidhardt F. C., VanBogelen R. A., Vaughn V. The genetics and regulation of heat-shock proteins. Annu Rev Genet. 1984;18:295–329. doi: 10.1146/annurev.ge.18.120184.001455. [DOI] [PubMed] [Google Scholar]
  24. Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
  25. Paek K. H., Walker G. C. Escherichia coli dnaK null mutants are inviable at high temperature. J Bacteriol. 1987 Jan;169(1):283–290. doi: 10.1128/jb.169.1.283-290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  27. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  28. Sherman M. Y., Goldberg A. L. Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria. J Bacteriol. 1991 Nov;173(22):7249–7256. doi: 10.1128/jb.173.22.7249-7256.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Straus D. B., Walter W. A., Gross C. A. Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 1988 Dec;2(12B):1851–1858. doi: 10.1101/gad.2.12b.1851. [DOI] [PubMed] [Google Scholar]
  30. Waxman L., Goldberg A. L. Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La). Science. 1986 Apr 25;232(4749):500–503. doi: 10.1126/science.2938257. [DOI] [PubMed] [Google Scholar]
  31. Zylicz M., Ang D., Liberek K., Georgopoulos C. Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins. EMBO J. 1989 May;8(5):1601–1608. doi: 10.1002/j.1460-2075.1989.tb03544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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