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Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli.

Jana B, Panja S, Saha S, Basu T - BMC Microbiol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani - 741 235, West Bengal, India. bimal_edu@rediffmail.com

ABSTRACT

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.

Conclusion: 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.

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Stability of sigma-32 in E. coli MPh42 cells. Pulse-chase and immunoprecipitation experiment was performed using 100 μCi/ml 35S-methionine and anti-sigma-32 antibody, as described in 'Methods'. Panel A: cells grown at 30°C in the presence of CCCP, panel B: control cells at 30°C, and panel C: cells submitted to 50°C. The numbers in the lanes signify the time of chasing in minutes.
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Figure 3: Stability of sigma-32 in E. coli MPh42 cells. Pulse-chase and immunoprecipitation experiment was performed using 100 μCi/ml 35S-methionine and anti-sigma-32 antibody, as described in 'Methods'. Panel A: cells grown at 30°C in the presence of CCCP, panel B: control cells at 30°C, and panel C: cells submitted to 50°C. The numbers in the lanes signify the time of chasing in minutes.

Mentions: At different intervals of growth in the presence of CCCP, when the rate of sigma-32 synthesis was measured by the pulse-label and immunoprecipitation experiment, no change in the rate with the time of cell growth was observed (fig. 2A); whereas in cells grown at 50°C, the rate had increased up to 5 min (fig. 2B), after which it declined. Therefore, the rise in cellular sigma-32 level and thereby induction of hsps in E. coli by CCCP treatment did not occur by the enhanced synthesis of sigma-32. This result also indicated that the CCCP could not denature the secondary structure present in sigma-32 mRNA and thus entry of the mRNA into the ribosome and consequent increase of translation had been prevented. On the other hand, when the sigma-32 stabilization was investigated with the help of pulse-chase and immunoprecipitation experiment, no change in sigma-32 band intensity had been observed in the CCCP-treated cells up to 4 minutes of chasing (fig. 3A); whereas in case of control cells, sigma-32 intensity had been almost halved in 2 minutes of chasing (fig. 3B), signifying stabilization of sigma-32 in cells by CCCP treatment. When checked, sigma-32 was also found to be stabilized in cells grown at 50°C (fig. 3C). The above results, therefore, implied clearly that for induction of hsps in the CCCP-treated cells, cellular level of sigma-32 had been increased, not by its increased rate of synthesis, but by its increased stabilization.


Mechanism of protonophores-mediated induction of heat-shock response in Escherichia coli.

Jana B, Panja S, Saha S, Basu T - BMC Microbiol. (2009)

Stability of sigma-32 in E. coli MPh42 cells. Pulse-chase and immunoprecipitation experiment was performed using 100 μCi/ml 35S-methionine and anti-sigma-32 antibody, as described in 'Methods'. Panel A: cells grown at 30°C in the presence of CCCP, panel B: control cells at 30°C, and panel C: cells submitted to 50°C. The numbers in the lanes signify the time of chasing in minutes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654656&req=5

Figure 3: Stability of sigma-32 in E. coli MPh42 cells. Pulse-chase and immunoprecipitation experiment was performed using 100 μCi/ml 35S-methionine and anti-sigma-32 antibody, as described in 'Methods'. Panel A: cells grown at 30°C in the presence of CCCP, panel B: control cells at 30°C, and panel C: cells submitted to 50°C. The numbers in the lanes signify the time of chasing in minutes.
Mentions: At different intervals of growth in the presence of CCCP, when the rate of sigma-32 synthesis was measured by the pulse-label and immunoprecipitation experiment, no change in the rate with the time of cell growth was observed (fig. 2A); whereas in cells grown at 50°C, the rate had increased up to 5 min (fig. 2B), after which it declined. Therefore, the rise in cellular sigma-32 level and thereby induction of hsps in E. coli by CCCP treatment did not occur by the enhanced synthesis of sigma-32. This result also indicated that the CCCP could not denature the secondary structure present in sigma-32 mRNA and thus entry of the mRNA into the ribosome and consequent increase of translation had been prevented. On the other hand, when the sigma-32 stabilization was investigated with the help of pulse-chase and immunoprecipitation experiment, no change in sigma-32 band intensity had been observed in the CCCP-treated cells up to 4 minutes of chasing (fig. 3A); whereas in case of control cells, sigma-32 intensity had been almost halved in 2 minutes of chasing (fig. 3B), signifying stabilization of sigma-32 in cells by CCCP treatment. When checked, sigma-32 was also found to be stabilized in cells grown at 50°C (fig. 3C). The above results, therefore, implied clearly that for induction of hsps in the CCCP-treated cells, cellular level of sigma-32 had been increased, not by its increased rate of synthesis, but by its increased stabilization.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani - 741 235, West Bengal, India. bimal_edu@rediffmail.com

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus