Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli.
BACKGROUND
Protonophores are the agents that dissipate the proton-motive-force (PMF) across E. coli plasma membrane. As the PMF is known to be an energy source for the translocation of membrane and periplasmic proteins after their initial syntheses in cell cytoplasm, protonophores therefore inhibit the translocation phenomenon. In addition, protonophores also induce heat-shock-like stress response in E. coli cell. In this study, our motivation was to investigate that how the protonophores-mediated phenomena like inhibition of protein translocation and induction of heat-shock proteins in E. coli were correlated.


RESULTS
Induction of heat-shock-like response in E. coli attained the maximum level after about 20 minutes of cell growth in the presence of a protonophore like carbonyl cyanide m-chloro phenylhydrazone (CCCP) or 2, 4-dinitrophenol (DNP). With induction, cellular level of the heat-shock regulator protein sigma-32 also increased. The increase in sigma-32 level was resulted solely from its stabilization, not from its increased synthesis. On the other hand, the protonophores inhibited the translocation of the periplasmic protein alkaline phosphatase (AP), resulting its accumulation in cell cytosol partly in aggregated and partly in dispersed form. On further cell growth, after withdrawal of the protonophores, the previously accumulated AP could not be translocated out; instead the AP-aggregate had been degraded perhaps by an induced heat-shock protease ClpP. Moreover, the non-translocated AP formed binary complex with the induced heat-shock chaperone DnaK and the excess cellular concentration of DnaK disallowed the induction of heat-shock response by the protonophores.


CONCLUSIONS
Our experimental results suggested that the protonophores-mediated accumulation and aggregation of membrane proteins (like AP) in cell cytosol had signaled the induction of heat-shock proteins in E. coli and the non-translocated protein aggregates were possibly degraded by an induced heat-shock protease ClpP. Moreover, the induction of heat-shock response occurred by the stabilization of sigma-32. As, normally the DnaK-bound sigma-32 was known to be degraded by the heat-shock protease FtsH, our experimental results further suggested that the engagement of DnaK with the non-translocated proteins (like AP) had made the sigma-32 free and stable.
Jana
Bimal
B
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani - 741 235, West Bengal, India. bimal_edu@rediffmail.com
Panja
Subrata
S
Saha
Swati
S
Basu
Tarakdas
T
D019297:2,4-Dinitrophenol; D000469:Alkaline Phosphatase / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry; D049071:Endopeptidase Clp / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects*; D029968:Escherichia coli Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D018869:Heat-Shock Response; D007476:Ionophores / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D018892:Proton-Motive Force / Q000187:drug effects*; D012808:Sigma Factor / Q000378:metabolism
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007476:Ionophores; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000469:Alkaline Phosphatase; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp; C063842:dnaK protein, E coli; D019297:2,4-Dinitrophenol