Procaine, a Local Anesthetic Interacting with the Cell Membrane, Inhibits the Processing of Precursor Forms of Periplasmic Proteins in Escherichia coli (original) (raw)
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Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli
Proceedings of the National Academy of Sciences, 1981
The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space. The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential. Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts. Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium. These results suggest a role for membrane potential in the secretion of periplasmic proteins. A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence...
European Journal of Biochemistry, 1984
Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally.
Conditions leading to secretion of a normally periplasmic protein in Escherichia coli
Journal of bacteriology, 1987
The phosphate-binding protein (PhoS) is a periplasmic protein which is part of the high-affinity phosphate transport system of Escherichia coli. Hyperproduction of PhoS in strains carrying a multicopy plasmid containing phoS led to partial secretion of the protein. By 6 h after transfer to phosphate-limiting medium, about 13% of the total newly synthesized PhoS was secreted to the medium. Kinetic studies demonstrated that this secretion consists of newly synthesized PhoS. This secretion occurs in PhoS-hyperproducer strains but not in a PhoS-overproducer strain. Another type of secretion concerning periplasmic PhoS was observed in both PhoS-hyperproducer and PhoS-overproducer strains. This mode of secretion depended upon the addition of phosphate to cells previously grown in phosphate-limiting medium.
Journal of Biological Chemistry, 1995
We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G.(1994) EMBO J. 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and ...
Preserving the membrane barrier for small molecules during bacterial protein translocation
Nature, 2011
Many proteins are translocated through the SecY channel in bacteria and archaea, and the related Sec61 channel in eukaryotes 1. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids 2. While the cytoplasmic cavity of the channel is empty, the extra-cellular cavity is filled with a short helix, the plug 2 , which moves out of the way during protein translocation 3,4. The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial 2,5-8. Here, we have addressed this issuein intact E. coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. In the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation the pore ring forms a gasket-like seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms suggests a universal mechanism by which the membrane barrier is maintained during protein translocation. Bacteria offer a unique opportunity to test the permeation of small molecules through the protein translocation channel, as the channel is located in the plasma membrane and is therefore accessible in intact cells. To test the permeability of the resting channel, we compared E. coli wild-type SecY, expected to be sealed, with a plug-deletion mutant(ΔP), which should be constitutively open(Fig. S1); although a new plug may form from neighboring polypeptide segments 9 , it likely blocks the channel only transiently 8. Wild-type and ΔP mutant SecY channels were expressed under an inducible promoter at about the same level as the endogenous protein (Fig. S2). Expression of the ΔP mutant caused only a moderate growth defect (Fig. S2). Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: