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. 1996 Jan 15;15(2):437–444.

Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease.

J E Laachouch 1, L Desmet 1, V Geuskens 1, R Grimaud 1, A Toussaint 1
PMCID: PMC449959  PMID: 8617219

Abstract

Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.

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Selected References

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

  1. Bernard P., Couturier M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J Mol Biol. 1992 Aug 5;226(3):735–745. doi: 10.1016/0022-2836(92)90629-x. [DOI] [PubMed] [Google Scholar]
  2. Bernard P., Kézdy K. E., Van Melderen L., Steyaert J., Wyns L., Pato M. L., Higgins P. N., Couturier M. The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. J Mol Biol. 1993 Dec 5;234(3):534–541. doi: 10.1006/jmbi.1993.1609. [DOI] [PubMed] [Google Scholar]
  3. CAMPBELL A. Sensitive mutants of bacteriophage lambda. Virology. 1961 May;14:22–32. doi: 10.1016/0042-6822(61)90128-3. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  5. Chen S., Roseman A. M., Hunter A. S., Wood S. P., Burston S. G., Ranson N. A., Clarke A. R., Saibil H. R. Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy. Nature. 1994 Sep 15;371(6494):261–264. doi: 10.1038/371261a0. [DOI] [PubMed] [Google Scholar]
  6. Cheng H. H., Muhlrad P. J., Hoyt M. A., Echols H. Cleavage of the cII protein of phage lambda by purified HflA protease: control of the switch between lysis and lysogeny. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7882–7886. doi: 10.1073/pnas.85.21.7882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Geuskens V., Mhammedi-Alaoui A., Desmet L., Toussaint A. Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease. EMBO J. 1992 Dec;11(13):5121–5127. doi: 10.1002/j.1460-2075.1992.tb05619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geuskens V., Vogel J. L., Grimaud R., Desmet L., Higgins N. P., Toussaint A. Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage. J Bacteriol. 1991 Oct;173(20):6578–6585. doi: 10.1128/jb.173.20.6578-6585.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gottesman S., Clark W. P., de Crecy-Lagard V., Maurizi M. R. ClpX, an alternative subunit for the ATP-dependent Clp protease of Escherichia coli. Sequence and in vivo activities. J Biol Chem. 1993 Oct 25;268(30):22618–22626. [PubMed] [Google Scholar]
  10. Gottesman S., Gottesman M., Shaw J. E., Pearson M. L. Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability. Cell. 1981 Apr;24(1):225–233. doi: 10.1016/0092-8674(81)90518-3. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Leach D., Symonds N. The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment of Tn3 conferring ampicillin resistance. Mol Gen Genet. 1979 May 4;172(2):179–184. doi: 10.1007/BF00268280. [DOI] [PubMed] [Google Scholar]
  15. Lehnherr H., Yarmolinsky M. B. Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3274–3277. doi: 10.1073/pnas.92.8.3274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levchenko I., Luo L., Baker T. A. Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev. 1995 Oct 1;9(19):2399–2408. doi: 10.1101/gad.9.19.2399. [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. Mhammedi-Alaoui A., Pato M., Gama M. J., Toussaint A. A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Mol Microbiol. 1994 Mar;11(6):1109–1116. doi: 10.1111/j.1365-2958.1994.tb00387.x. [DOI] [PubMed] [Google Scholar]
  19. Mizusawa S., Gottesman S. Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein. Proc Natl Acad Sci U S A. 1983 Jan;80(2):358–362. doi: 10.1073/pnas.80.2.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peters J. M. Proteasomes: protein degradation machines of the cell. Trends Biochem Sci. 1994 Sep;19(9):377–382. doi: 10.1016/0968-0004(94)90115-5. [DOI] [PubMed] [Google Scholar]
  21. Roberts J. W., Roberts C. W. Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc Natl Acad Sci U S A. 1975 Jan;72(1):147–151. doi: 10.1073/pnas.72.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shapiro J. A. A role for the Clp protease in activating Mu-mediated DNA rearrangements. J Bacteriol. 1993 May;175(9):2625–2631. doi: 10.1128/jb.175.9.2625-2631.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Singh S. K., Maurizi M. R. Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli. J Biol Chem. 1994 Nov 25;269(47):29537–29545. [PubMed] [Google Scholar]
  25. Squires C., Squires C. L. The Clp proteins: proteolysis regulators or molecular chaperones? J Bacteriol. 1992 Feb;174(4):1081–1085. doi: 10.1128/jb.174.4.1081-1085.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thompson M. W., Maurizi M. R. Activity and specificity of Escherichia coli ClpAP protease in cleaving model peptide substrates. J Biol Chem. 1994 Jul 8;269(27):18201–18208. [PubMed] [Google Scholar]
  27. Thompson M. W., Singh S. K., Maurizi M. R. Processive degradation of proteins by the ATP-dependent Clp protease from Escherichia coli. Requirement for the multiple array of active sites in ClpP but not ATP hydrolysis. J Biol Chem. 1994 Jul 8;269(27):18209–18215. [PubMed] [Google Scholar]
  28. Tobias J. W., Shrader T. E., Rocap G., Varshavsky A. The N-end rule in bacteria. Science. 1991 Nov 29;254(5036):1374–1377. doi: 10.1126/science.1962196. [DOI] [PubMed] [Google Scholar]
  29. Torres-Cabassa A. S., Gottesman S. Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J Bacteriol. 1987 Mar;169(3):981–989. doi: 10.1128/jb.169.3.981-989.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Van Melderen L., Bernard P., Couturier M. Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria. Mol Microbiol. 1994 Mar;11(6):1151–1157. doi: 10.1111/j.1365-2958.1994.tb00391.x. [DOI] [PubMed] [Google Scholar]
  31. Vogel J. L., Li Z. J., Howe M. M., Toussaint A., Higgins N. P. Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain. J Bacteriol. 1991 Oct;173(20):6568–6577. doi: 10.1128/jb.173.20.6568-6577.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Wickner S., Gottesman S., Skowyra D., Hoskins J., McKenney K., Maurizi M. R. A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12218–12222. doi: 10.1073/pnas.91.25.12218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wojtkowiak D., Georgopoulos C., Zylicz M. Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli. J Biol Chem. 1993 Oct 25;268(30):22609–22617. [PubMed] [Google Scholar]
  35. Yoo S. J., Seol J. H., Kang M. S., Ha D. B., Chung C. H. clpX encoding an alternative ATP-binding subunit of protease Ti (Clp) can be expressed independently from clpP in Escherichia coli. Biochem Biophys Res Commun. 1994 Sep 15;203(2):798–804. doi: 10.1006/bbrc.1994.2253. [DOI] [PubMed] [Google Scholar]
  36. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

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