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The C-terminal fragment of colicin, a protein that is highly soluble in aqueous solution, is spontaneously and irreversibly inserted into the membranes of mammalian cells, which are locally permeabilized by a transmembrane voltage increase. Insertion is detected by immunodetection. This is obtained by mixing the protein with electropermeabilized cells. The same result is observed by pulsing the colicin/cell mixture. Electroinsertion is therefore obtained for the first time with a multi-fragment spanning protein. The cell viability is not affected beyond the effect of electropermeabilization. A train of low voltage repetitive transmembrane modulation, which cannot trigger membrane permeabilization, is applied a day after the electroinsertion. This induces no effect on unmodified cells but triggers the lysis of cells in which colicin has been inserted by the first electropulsation. The low-level electrical treatment is high enough to trigger the voltage gated opening of colicin and to induce the associated toxicity. A transmembrane configuration of colicin is therefore obtained by electroinsertion. The toxic effect of their voltage gating is only obtained when a critical number of voltage gated channels are activated. [ABSTRACT FROM AUTHOR]

Inhibition of lipid synthesis in cerulenin-treated cells or in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase after glycerol deprivation. results in a marked decrease of insertion of LamB protein into the outer membrane. No lambda receptor was found in any other cell compartment or in the medium under these conditions. The LamB protein synthesis was inhibited by about 70%, in the absence of lipid synthesis. The residual 30% protein produced during inhibition of fatty-acid or phospholipid synthesis, was probably incorporated into the outer membrane since no Further incorporation was observed after resumption of these syntheses. Besides OmpF and OmpC protein [Bocquet-Pagés, C.. Lazdunski. C., and Lazdunski1 A. (1981) Eur. f. Biochem 105 - 111]. at least four other proteins of the outer membrane are also subject to alteration of levels in the absence of lipid synthesis. Under these conditions the uptake of maltose, like the uptake of 5'AMP [Bocquet-Pagés. C., Lazdunski. C.. and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105- 111]. was inhibited as much as 60%. These results are discussed with regard to the biosynthesis and assembly of the outer membrane proteins. [ABSTRACT FROM AUTHOR]

DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a ‘STOP-Shine-Dalgarno-sequence-ATG’ cassette encoding a termination codon, a Shine- Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH2-terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X-114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1–3 of the 204-amino-acid-residue colicin pore-forming domain (containing 10 α-helices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH-terminal domain. [ABSTRACT FROM AUTHOR]

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N-terminal domain of colicins is involved in the import process. The N-terminal domains of colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N-terminal domain of all group A colicins, the ‘TolA box’, was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N-terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N-terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N-terminal domain interact with TolA and TolB. The colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB. [ABSTRACT FROM AUTHOR]

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins. The N-terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B). Producing ceils became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB. Hence, this Interaction must play an important role in colicin E3 translocation. [ABSTRACT FROM AUTHOR]

Pore-forming colicins are a family of protein toxins (Mr 40-70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called 'Tol' proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. TheX-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of... [ABSTRACT FROM AUTHOR]

Colicin A is a pore-forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c1 (pc1) as well as the first 167 amino acids of cytochrome b2 (pb2) were fused to the pore-forming domain of colicin A (pfColA). Both hybrid proteins (pc1-pfColA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore-forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome c1 presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c, pre-sequence moiety of pc1-pfColA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1-pfColA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (ii) this channel activity was inhibited within the membrane by the immunity protein. [ABSTRACT FROM AUTHOR]

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins. [ABSTRACT FROM AUTHOR]

Spheroplasts, prepared according to an efficient procedure which is described, secrete a smaller protein fraction of the total cell content than that secreted by intact cells. However, more proteins are found associated to the cell envelopes derived from these spheroplasts than to those derived from intact cells. This result was confirmed when we measured, by specific immunoprecipitation, the amount of secreted and unsecreted alkaline phosphatase. Whereas at any time, at 30 °C, in intact cells 99.6%, of the alkaline phosphatase produced is secreted and found in the periplasm [see A. Torriani (1968) J. Bacteriol. 96, 1200–1207], only 40% was secreted by our spheroplast preparation. We suggest that partial depletion of outer-membrane-bound protease (or proteases), involved in processing of precursors of secreted protein, is responsible for the significant defect of secretion of precursors which then remain membrane-bound. Such an experimental protocol has been carried out at various temperatures and the secretion of alkaline phosphatase was assayed. This secretion was found to slightly vary above 22 °C whereas it dramatically decreased below 22 °C. Practically no phosphatase was secreted at 13 °C. However, the synthesis of alkaline phosphatase was not differentially altered at low temperatures compared to total proteins. The inactive presumed precursor of this enzyme which was then not secreted was found associated to the cell envelope. Similar results have been observed in intact cells above 22 °C where only the energy of protein synthesis (ΔE = 79 k J), which causes the elongation of nascent chains, is used for protein secretion; below 22 °C, in the temperature range of the disorder-order transition of the lipid paraffin chains associated to membrane, a ΔE of 125 kJ is required for secretion of alkaline phosphatase. A ΔE of 146 kJ was found for this process to occur in the same temperature range in spheroplasts. When oleate was incorporated into the membrane of strain K 1059, an unsaturated fatty acid auxotroph, the same result as with wild-type strains was observed, but a decrease in secretion at high temperatures was induced by this modification of fatty acid composition of the membrane. When elaidate instead of oleate was incorporated into K 1059 membrane, the fall in alkaline phosphatase secretion occurred at 33 °C instead of 22 °C, which indicates that the physical state of the lipid phase is really involved in the secretion process. A hypothetical model for secretion of nascent periplasmic proteins, based on the data reported, is presented. [ABSTRACT FROM AUTHOR]