Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel mscL - PubMed (original) (raw)

Comparative Study

Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel mscL

C Berrier et al. J Bacteriol. 2000 Jan.

Abstract

Upon osmotic downshock, a few cytoplasmic proteins, including thioredoxin, elongation factor Tu (EF-Tu), and DnaK, are released from Tris-EDTA-treated Escherichia coli cells by an unknown mechanism. We have shown previously that deletion of mscL, the gene coding for the mechanosensitive channel of the plasma membrane with the highest conductance, prevents the release of thioredoxin. We confirm and extend the implication of MscL in this process by showing that the release of EF-Tu and DnaK is severely impaired in MscL-deficient strains. Release of these proteins is not observed in the absence of a Tris-EDTA treatment which disrupts the outer membrane, indicating that, in intact cells, they are transferred to the periplasm upon shock, presumably through the MscL channel.

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Figures

FIG. 1

FIG. 1

Shock-induced release of EF-Tu. Cells were resuspended at _A_650 = 5 in 10 mM Tris (pH 7.6) and 20% (wt/vol) sucrose, without (A) or with (B and C) 2.5 mM EDTA for 10 min at room temperature. Isosmotic dilution and hyposmotic shock were performed by diluting 200 μl of the suspension fivefold with the same buffer or distilled water. The suspension was centrifuged, and the presence of EF-Tu in the pellet and in the supernatant was revealed by immunoblotting as previously described (1), with rabbit antibody against EF-Tu. The pellet was suspended and boiled 4 min in 100 μl of electrophoresis buffer (50 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 0.2% bromophenol blue, 10% glycerol, 1% β-mercaptoethanol) and a 4-μl aliquot was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 200-μl aliquot of the supernatant was incubated overnight with 20% trichloroacetic acid and centrifuged. The precipitated proteins were suspended in 20 μl of electrophoresis buffer and boiled for 4 min, and 4 μl was subjected to electrophoresis. For determination of the total EF-Tu cell content, 1 ml of cells (_A_650 = 1) was centrifuged, the pellet was suspended in 100 μl of electrophoresis buffer, and a 4-μl sample was subjected to electrophoresis. (A) EF-Tu is not released by cells that are not treated with EDTA. Wild-type cells were suspended in Tris buffer and diluted fivefold with the same buffer or distilled water. Lane 1, total EF-Tu cell content before shock. The remaining lanes show EF-Tu in the pellet (P) and supernatant (S) after isosmotic dilution (lane 2), after osmotic shock (lane 3), and after osmotic shock in the presence of 1 mM gadolinium (lane 4). (B) Release of EF-Tu after EDTA treatment. Wild-type cells were suspended in Tris buffer containing 2.5 mM EDTA and diluted fivefold with the same buffer or distilled water. Lanes 1 to 4 are as above. (C) Release of EF-Tu is impaired in MscL-deficient cells. Wild-type (WT), MscL-deficient (MscL−), and restored (R) cells were EDTA treated and subjected to an osmotic shock. EF-Tu in the pellet and supernatant was revealed by immunoblotting. (D) Quantification by densitometry of the protein bands shown in C.

FIG. 2

FIG. 2

Shock-induced release of DnaK. Experiments were performed as described for Fig. 1, and the presence of DnaK was detected by immunoblotting as previously described (1), with rabbit antibody against DnaK. (A) DnaK is not released by cells that are not treated with EDTA. Wild-type cells were suspended in Tris buffer and diluted fivefold with the same buffer or distilled water. Lane 1, total EF-Tu cell content before shock. The remaining lanes show EF-Tu in the pellet (P) and supernatant (S) after isosmotic dilution (lane 2), after osmotic shock (lane 3), and after osmotic shock in the presence of 1 mM gadolinium (lane 4). (B) Release of DnaK after EDTA treatment. Wild-type cells were suspended in Tris buffer containing 2.5 mM EDTA and diluted fivefold with the same buffer or distilled water. Lanes 1 to 4 are as above. (C) Release of DnaK is impaired in MscL-deficient cells. Wild-type (WT), MscL-deficient (MscL−), and restored (R) cells were EDTA treated and subjected to an osmotic shock. DnaK in the pellet and supernatant was revealed by immunoblotting. (D) Quantification by densitometry of the protein bands shown in C.

FIG. 3

FIG. 3

Release of osmolytes and proteins during osmotic downshock. Osmotic downshock triggers the opening mechanosensitive channels in the plasma membrane. Osmolytes are released through MscL, MscS, and possibly MscM to the periplasm. Osmolytes can then diffuse out of the cell through porins. Cytoplasmic proteins such as thioredoxin, EF-Tu, and DnaK, are excreted mostly through MscL, the channel with the largest pore, to the periplasm, where they remain trapped because their size does not allow them to cross the outer membrane.

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