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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.

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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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A. Formation of AP-DnaK binary complex in CCCP-treated cells. Log phase cells, in phosphate-free MOPS medium, were labeled with 35S-methionine (30 μCi/ml) for 30 min at 30°C in presence of 50 μM CCCP. 1 ml labeled cells was chilled, centrifuged and resuspended in 200 μl Tris buffer (30 mM, pH 8.0) containing 20% sucrose, 10 mM EDTA (pH 8.0), 1 mg/ml lysozyme and the cell suspension was kept at 4°C for 10 min. 1 ml lysis solution [50 mM Tris (pH 8.0), 40 mM NaCl and 0.1% Tween 20] was added to the cell suspension and placed on ice for 30 min; NaCl was then added to a final concentration of 0.2 M and the cell lysate was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was first immunoprecipitated with anti-DnaK antibody. The immunocomplex was washed with above lysis solution containing 0.2 M NaCl, suspended in 100 μl Tris (pH 7.4), heated at 100°C for 3 min and finally immunoprecipitated with anti-AP antibody. The immunoprecipitate was run in 12% SDS-polyacrylamide gel and finally phosphorimaged. Lane a: CCCP-treated cell; lane b: control cell. B. State of GroEL induction in cells containing excess DnaK. Transformed cells were primarily grown up to log phase (~1.5 × 108 cells/ml) at 30°C in MOPS medium. 1 mM IPTG was then added and growth was allowed for another 30 min (to induce DnaK). The cells were transferred to methionine-free MOPS medium, grown further in presence of 50 μM CCCP for 20 min and then labeled with 35S-metthionine (30 μCi/ml) for 10 min. Parallel experiment was done for untransformed cells also. Cell extracts were then prepared by boiling with SDBME buffer. Equal amount of protein extract from both transformed and untransformed cells, as estimated by Bradford method, was subjected to immunoprecipitation using anti-GroEL antibody. The immunoprecipitate was run in SDS-polyacylamide gel and phosphoroimaged. Lane a: untransformed cell; lane b: transformed cell.
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Figure 7: A. Formation of AP-DnaK binary complex in CCCP-treated cells. Log phase cells, in phosphate-free MOPS medium, were labeled with 35S-methionine (30 μCi/ml) for 30 min at 30°C in presence of 50 μM CCCP. 1 ml labeled cells was chilled, centrifuged and resuspended in 200 μl Tris buffer (30 mM, pH 8.0) containing 20% sucrose, 10 mM EDTA (pH 8.0), 1 mg/ml lysozyme and the cell suspension was kept at 4°C for 10 min. 1 ml lysis solution [50 mM Tris (pH 8.0), 40 mM NaCl and 0.1% Tween 20] was added to the cell suspension and placed on ice for 30 min; NaCl was then added to a final concentration of 0.2 M and the cell lysate was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was first immunoprecipitated with anti-DnaK antibody. The immunocomplex was washed with above lysis solution containing 0.2 M NaCl, suspended in 100 μl Tris (pH 7.4), heated at 100°C for 3 min and finally immunoprecipitated with anti-AP antibody. The immunoprecipitate was run in 12% SDS-polyacrylamide gel and finally phosphorimaged. Lane a: CCCP-treated cell; lane b: control cell. B. State of GroEL induction in cells containing excess DnaK. Transformed cells were primarily grown up to log phase (~1.5 × 108 cells/ml) at 30°C in MOPS medium. 1 mM IPTG was then added and growth was allowed for another 30 min (to induce DnaK). The cells were transferred to methionine-free MOPS medium, grown further in presence of 50 μM CCCP for 20 min and then labeled with 35S-metthionine (30 μCi/ml) for 10 min. Parallel experiment was done for untransformed cells also. Cell extracts were then prepared by boiling with SDBME buffer. Equal amount of protein extract from both transformed and untransformed cells, as estimated by Bradford method, was subjected to immunoprecipitation using anti-GroEL antibody. The immunoprecipitate was run in SDS-polyacylamide gel and phosphoroimaged. Lane a: untransformed cell; lane b: transformed cell.

Mentions: When the cell extract of AP-induced culture was subjected to two-step immunoprecipitation study using anti-DnaK and anti-AP antibodies serially, the final immunoprecipitate of the CCCP-treated cells, in contrast to that of the control cells, had contained AP in addition to the DnaK protein (fig. 7A). This clearly signified that the first immunoprecipitate with anti-DnaK antibody had certainly contained AP i.e., the non-translocated AP in the CCCP-treated cells was present in cell cytosol as a binary complex form with DnaK. This result justified the fact of sigma-32 stabilization in the protonophores-treated cells as – the non-translocated proteins had signaled DnaK/J to bind with them, finally freeing and so stabilizing sigma-32. This was further confirmed by our observation that in the cells containing over-expressed DnaK, the CCCP could not trigger the induction of hsps (fig. 7B); this was because the sigma-32 could not be freed from the DnaK to bind with the RNA polymarease, due to the excess cellular pool of DnaK protein. For this study, cells of E. coli MPh42 were transformed with plasmid pET vector containing dnaK gene and the DnaK protein was over-expressed by using 1 mM IPTG in the MOPS growth medium. When such excess DnaK-containing cells were subsequently grown in the presence of 50 μM CCCP and the cell extract was immunoprecipitated using anti-GroEL antibody, no induction of GroEL had been observed in the CCCP-treated transformed cells (lane b, fig. 7B); whereas the induction had occurred in the CCCP-treated untransformed cells (lane a, fig. 7B). This result implied that no induction of hsps had taken place in the CCCP-treated cells having excess amount of DnaK chaperone.


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

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

A. Formation of AP-DnaK binary complex in CCCP-treated cells. Log phase cells, in phosphate-free MOPS medium, were labeled with 35S-methionine (30 μCi/ml) for 30 min at 30°C in presence of 50 μM CCCP. 1 ml labeled cells was chilled, centrifuged and resuspended in 200 μl Tris buffer (30 mM, pH 8.0) containing 20% sucrose, 10 mM EDTA (pH 8.0), 1 mg/ml lysozyme and the cell suspension was kept at 4°C for 10 min. 1 ml lysis solution [50 mM Tris (pH 8.0), 40 mM NaCl and 0.1% Tween 20] was added to the cell suspension and placed on ice for 30 min; NaCl was then added to a final concentration of 0.2 M and the cell lysate was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was first immunoprecipitated with anti-DnaK antibody. The immunocomplex was washed with above lysis solution containing 0.2 M NaCl, suspended in 100 μl Tris (pH 7.4), heated at 100°C for 3 min and finally immunoprecipitated with anti-AP antibody. The immunoprecipitate was run in 12% SDS-polyacrylamide gel and finally phosphorimaged. Lane a: CCCP-treated cell; lane b: control cell. B. State of GroEL induction in cells containing excess DnaK. Transformed cells were primarily grown up to log phase (~1.5 × 108 cells/ml) at 30°C in MOPS medium. 1 mM IPTG was then added and growth was allowed for another 30 min (to induce DnaK). The cells were transferred to methionine-free MOPS medium, grown further in presence of 50 μM CCCP for 20 min and then labeled with 35S-metthionine (30 μCi/ml) for 10 min. Parallel experiment was done for untransformed cells also. Cell extracts were then prepared by boiling with SDBME buffer. Equal amount of protein extract from both transformed and untransformed cells, as estimated by Bradford method, was subjected to immunoprecipitation using anti-GroEL antibody. The immunoprecipitate was run in SDS-polyacylamide gel and phosphoroimaged. Lane a: untransformed cell; lane b: transformed cell.
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Figure 7: A. Formation of AP-DnaK binary complex in CCCP-treated cells. Log phase cells, in phosphate-free MOPS medium, were labeled with 35S-methionine (30 μCi/ml) for 30 min at 30°C in presence of 50 μM CCCP. 1 ml labeled cells was chilled, centrifuged and resuspended in 200 μl Tris buffer (30 mM, pH 8.0) containing 20% sucrose, 10 mM EDTA (pH 8.0), 1 mg/ml lysozyme and the cell suspension was kept at 4°C for 10 min. 1 ml lysis solution [50 mM Tris (pH 8.0), 40 mM NaCl and 0.1% Tween 20] was added to the cell suspension and placed on ice for 30 min; NaCl was then added to a final concentration of 0.2 M and the cell lysate was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was first immunoprecipitated with anti-DnaK antibody. The immunocomplex was washed with above lysis solution containing 0.2 M NaCl, suspended in 100 μl Tris (pH 7.4), heated at 100°C for 3 min and finally immunoprecipitated with anti-AP antibody. The immunoprecipitate was run in 12% SDS-polyacrylamide gel and finally phosphorimaged. Lane a: CCCP-treated cell; lane b: control cell. B. State of GroEL induction in cells containing excess DnaK. Transformed cells were primarily grown up to log phase (~1.5 × 108 cells/ml) at 30°C in MOPS medium. 1 mM IPTG was then added and growth was allowed for another 30 min (to induce DnaK). The cells were transferred to methionine-free MOPS medium, grown further in presence of 50 μM CCCP for 20 min and then labeled with 35S-metthionine (30 μCi/ml) for 10 min. Parallel experiment was done for untransformed cells also. Cell extracts were then prepared by boiling with SDBME buffer. Equal amount of protein extract from both transformed and untransformed cells, as estimated by Bradford method, was subjected to immunoprecipitation using anti-GroEL antibody. The immunoprecipitate was run in SDS-polyacylamide gel and phosphoroimaged. Lane a: untransformed cell; lane b: transformed cell.
Mentions: When the cell extract of AP-induced culture was subjected to two-step immunoprecipitation study using anti-DnaK and anti-AP antibodies serially, the final immunoprecipitate of the CCCP-treated cells, in contrast to that of the control cells, had contained AP in addition to the DnaK protein (fig. 7A). This clearly signified that the first immunoprecipitate with anti-DnaK antibody had certainly contained AP i.e., the non-translocated AP in the CCCP-treated cells was present in cell cytosol as a binary complex form with DnaK. This result justified the fact of sigma-32 stabilization in the protonophores-treated cells as – the non-translocated proteins had signaled DnaK/J to bind with them, finally freeing and so stabilizing sigma-32. This was further confirmed by our observation that in the cells containing over-expressed DnaK, the CCCP could not trigger the induction of hsps (fig. 7B); this was because the sigma-32 could not be freed from the DnaK to bind with the RNA polymarease, due to the excess cellular pool of DnaK protein. For this study, cells of E. coli MPh42 were transformed with plasmid pET vector containing dnaK gene and the DnaK protein was over-expressed by using 1 mM IPTG in the MOPS growth medium. When such excess DnaK-containing cells were subsequently grown in the presence of 50 μM CCCP and the cell extract was immunoprecipitated using anti-GroEL antibody, no induction of GroEL had been observed in the CCCP-treated transformed cells (lane b, fig. 7B); whereas the induction had occurred in the CCCP-treated untransformed cells (lane a, fig. 7B). This result implied that no induction of hsps had taken place in the CCCP-treated cells having excess amount of DnaK chaperone.

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