1887

Abstract

Submission of wild-type to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26470-0
2004-01-01
2020-04-01
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/1/mic1500247.html?itemId=/content/journal/micro/10.1099/mic.0.26470-0&mimeType=html&fmt=ahah

References

  1. Allen S. P., Polazzi J. O., Gierse J. K., Easton A. M.. 1992; Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli. J Bacteriol174:6938–6947
    [Google Scholar]
  2. Ang D., Chandrasekhar G. N., Z˙ylicz M., Georgopoulos C.. 1986; Escherichia coli grpE gene codes heat shock protein B25.3, essential for both λ DNA replication at all temperatures and host growth at high temperature. J Bacteriol167:25–29
    [Google Scholar]
  3. Arsène F., Tomoyasu T., Bukau B.. 2000; The heat shock response of Escherichia coli. Int J Food Microbiol55:3–9[CrossRef]
    [Google Scholar]
  4. Błaszczak A., Georgopoulos C., Liberek K.. 1999; On the mechanism of FtsH-dependent degradation of σ32 transcriptional regulator of Escherichia coli and the role of the DnaK chaperone machine. Mol Microbiol31:157–166[CrossRef]
    [Google Scholar]
  5. Bradford M. M.. 1976; A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem72:248–254[CrossRef]
    [Google Scholar]
  6. Bukau B., Horwich A. L.. 1998; The Hsp70 and Hsp60 chaperone machines. Cell92:351–366[CrossRef]
    [Google Scholar]
  7. Chuang S. E., Blattner F. R.. 1993; Characterization of twenty-six new heat shock genes of Eschericha coli. J Bacteriol175:5242–5252
    [Google Scholar]
  8. Connolly L., Yura T., Gross C.. 1999; Autoregulation of the heat shock response in procaryotes. In Molecular Chaperones and Folding Catalysts. Regulation, Cellular Function and Mechanism pp.13–33Edited by Bukau B.. Amsterdam: Harwood Academic Publishers;
    [Google Scholar]
  9. Delaney J. M.. 1990; A grpE mutant of Escherichia coli is more resistant to heat shock than wild type. J Gen Microbiol136:797–801[CrossRef]
    [Google Scholar]
  10. Diamant S., Ben-Zwi A. P., Bukau B., Goloubinoff P.. 2000; Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery. J Biol Chem275:21107–21113[CrossRef]
    [Google Scholar]
  11. Gamer J., Multhaup G., Tomoyasu T., McCarty J. S., Rüdiger S., Schönfeld H. J., Schirra C., Bujard H., Bukau B.. 1996; A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor σ32. EMBO J15:607–617
    [Google Scholar]
  12. Georgopoulos C., Ang D., Liberek K., Z˙ylicz M.. 1990; Properties of Escherichia coli heat shock proteins and their role in bacteriophage λ growth. In Stress Proteins in Biology and Medicine pp.191–221 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  13. Goloubinoff P., Mogk A., Ben-Zwi A. B., Tomoyasu T., Bukau B.. 1999; Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc Natl Acad Sci U S A96:13732–13737[CrossRef]
    [Google Scholar]
  14. Grimshaw J. P., Jelesarov I., Siegenthaler R. K., Christen P.. 2003; Thermosensor action of GrpE. The DnaK chaperone system at heat shock temperatures. J Biol Chem278:19048–19053[CrossRef]
    [Google Scholar]
  15. Harrison C. J., Hayer-Hartl M., Di Liberto M., Hartl F. U., Kuriyan J.. 1997; Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science276:431–435[CrossRef]
    [Google Scholar]
  16. Henderson I., Garcia-Junceda E., Liu K. K. C., Chen Y. L., Shen G. J., Wong C. H.. 1994; Cloning, overexpression and isolation of the type II FDP aldolase from E. coli specificity study and synthetic application. . Bioorg Med Chem2:837–843[CrossRef]
    [Google Scholar]
  17. Hesterkamp T., Hauser S., Lützke H., Bukau B.. 1996; Escherichia coli trigger factor is a prolylisomerase that associates with nascent polypeptide chains. Proc Natl Acad Sci U S A93:4437–4441[CrossRef]
    [Google Scholar]
  18. Huang H. C., Sherman M. Y., Kandror O., Goldberg A. L.. 2001; The molecular chaperone DnaJ is required for degradation of a soluble abnormal protein in Escherichia coli. J Biol Chem276:3920–3928[CrossRef]
    [Google Scholar]
  19. Kanemori M., Nishihara K., Yanagi H., Yura T.. 1997; Synergistic roles of HslVU and other ATP-dependent proteases in controling in vivo turnover of σ32 and abnormal proteins in Escherichia coli. J Bacteriol179:7219–7225
    [Google Scholar]
  20. Kędzierska S., Staniszewska M., Węgrzyn A., Taylor A.. 1999; The role of DnaK/DnaJ and GroEL/GroES systems in removal of endogenous proteins aggregated by heat-shock from Escherichia coli. FEBS Lett446:331–337[CrossRef]
    [Google Scholar]
  21. Kędzierska S., Jezierski G., Taylor A.. 2001; DnaK/DnaJ chaperone system reactivates endogenous E. coli thermostable FBP aldolase in vivo and in vitro; the effect is enhanced by GroE heat-shock proteins. Cell Stress Chaperones6:29–37[CrossRef]
    [Google Scholar]
  22. Kitagawa M., Miyakawa M., Matsumura Y., Tsuchido T.. 2002; E. coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. Eur J Biochem269:2907–2917[CrossRef]
    [Google Scholar]
  23. Kucharczyk K., Laskowska E., Taylor A.. 1991; Response of Escherichia coli cell membranes to induction of λcI857 prophage by heat shock. Mol Microbiol5:2935–2945[CrossRef]
    [Google Scholar]
  24. Kuczyńska-Wiśnik D., Laskowska E., Taylor A.. 2001; Transcription of the ibpB gene is under control of σ32- and σ54-promoters; a third regulon of heat-shock response. Biochem Biophys Res Commun284:57–64[CrossRef]
    [Google Scholar]
  25. Kuczyńska-Wiśnik D., Kędzierska S., Matuszewska E., Lund P., Taylor A., Lipińska B., Laskowska E.. 2002; The E. coli small heat shock proteins IbpA and IbpB prevent the aggregation of endogenous proteins denatured in vivo during extreme heat shock. Microbiology148:1757–1765
    [Google Scholar]
  26. Laemmli U. K.. 1970; Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  27. Laskowska E., Kuczyńska-Wiśnik D., Skórko-Glonek J., Taylor A.. 1996a; Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro. Mol Microbiol22:555–571[CrossRef]
    [Google Scholar]
  28. Laskowska E., Wawrzynów A., Taylor A.. 1996b; IbpA and IbpB, the new heat-shock proteins, bind to endogenous Escherichia coli proteins aggregated intracellularly by heat shock. Biochimie78:117–122[CrossRef]
    [Google Scholar]
  29. Laufen T., Mayer M. P., Beisel C., Klostermeier D., Mogk A., Reinstein J., Bukau B.. 1999; Mechanism of regulation of Hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A96:5452–5457[CrossRef]
    [Google Scholar]
  30. Liberek K., Marszałek J., Ang D., Georgopoulos C., Z˙ylicz M.. 1991; Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Acad Natl Sci U S A88:2874–2878[CrossRef]
    [Google Scholar]
  31. Liberek K., Wall D., Georgopoulos C.. 1995; The DnaJ chaperone catalytically activates the DnaK chaperone to preferentially bind the σ32 heat shock transcriptional regulator. Proc Natl Acad Sci U S A92:6224–6228[CrossRef]
    [Google Scholar]
  32. Mally A., Witt S. N.. 2001; GrpE accelerates peptide binding and release from the high affinity state of DnaK. Nat Struct Biol8:254–257[CrossRef]
    [Google Scholar]
  33. Mitraki A., King J.. 1989; Protein folding intermediates and inclusion body formation. Biotechnology7:690–697[CrossRef]
    [Google Scholar]
  34. Mogk A., Tomoyasu T., Goloubinoff P., Rüdiger S., Röder D., Langen H., Bukau B.. 1999; Identification of thermolabile Escherichia coli proteins: reversion of aggregation by DnaK and ClpB. EMBO J18:6934–6949[CrossRef]
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  36. Schlieker C., Bukau B., Mogk A.. 2002; Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implication for their applicability in biotechnology. J Biotechnol96:13–21[CrossRef]
    [Google Scholar]
  37. Shearstone J. R., Baneyx F.. 1999; Biochemical characterization of the small heat shock protein IbpB from Escherichia coli. J Biol Chem274:9937–9945[CrossRef]
    [Google Scholar]
  38. Sherman M. Y., Goldberg A. L.. 1992; Involvement of chaperonin DnaK in the rapid degradation of a mutant protein in Escherichia coli. EMBO J11:71–77
    [Google Scholar]
  39. Spurr A. R.. 1969; A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res26:31–34[CrossRef]
    [Google Scholar]
  40. Straus D. B., Walter W. A., Gross C. A.. 1988; Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev2:1841–1858
    [Google Scholar]
  41. Strom M. S., Nunn D. N., Lory S.. 1993; A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc Natl Acad Sci U S A90:2404–2408[CrossRef]
    [Google Scholar]
  42. Teter S. A., Houry W. A., Ang D., Tradler T., Rockabrand T., Fischer G., Blum P., Georgopoulos C., Hartl F. U.. 1999; Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains. Cell97:755–765[CrossRef]
    [Google Scholar]
  43. Thomas J. G., Baneyx F.. 1998; Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: Comparison with ClpA, ClpB, and HtpGin vivo. J Bacteriol180:5165–5172
    [Google Scholar]
  44. Thomas J. G., Baneyx F.. 2000; ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells. Mol Microbiol36:1360–1370
    [Google Scholar]
  45. Veinger L., Diamant S., Buchner J., Goloubinoff P.. 1998; The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network. J Biol Chem273:11032–11037[CrossRef]
    [Google Scholar]
  46. Weibel E. R., Bolender R.. 1973; Stereological technique for electron microscopic morphometry. In Principles and Techniques of Electron Microscopy pp.237–353Edited by Hayat M.. New York: Van Nostrand Reynolds;
    [Google Scholar]
  47. Wu B., Ang D., Snavely M., Georgopoulos C.. 1994; Isolation and characterisation of point mutations in the Escherichia coligrpE heat shock gene. . J Bacteriol176:6965–6973
    [Google Scholar]
  48. Yoshimune K., Yoshimura T., Nakayama T., Nishino T., Esaki N.. 2002; Hsa62, Hsc56, and GrpE, the third Hsp70 chaperone system of E. coli. Biochem Biophys Res Comm293:1389–1395[CrossRef]
    [Google Scholar]
  49. Yura T., Tobe T., Ito K., Osawa T.. 1984; Heat shock regulatory gene htpR of Escherichia coli is required for growth at high temperature but is dispensable at low temperature. Proc Natl Acad Sci U S A81:6803–6807[CrossRef]
    [Google Scholar]
  50. Zolkiewski M.. 1999; ClpB cooperates with DnaK, DnaJ and GrpE in suppressing protein aggregation. J Biol Chem274:28083–28086[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26470-0
Loading
/content/journal/micro/10.1099/mic.0.26470-0
Loading

Data & Media loading...

Most cited this month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error