Majdalani, N and Ippen-Ihler, K (1996) Membrane insertion of the F-pilin subunit is Sec independent but requires leader peptidase B and the proton motive force. J. Bacteriol. 178:3742-7
F pilin is the subunit required for the assembly of conjugative pili on the cell surface of Escherichia coli carrying the F plasmid. Maturation of the F-pilin precursor, propilin, involves three F plasmid transfer products: TraA, the propilin precursor; TraQ, which promotes efficient propilin processing; and TraX, which is required for acetylation of the amino terminus of the 7-kDa pilin polypeptide. The mature pilin begins at amino acid 52 of the TraA propilin sequence. We performed experiments to determine the involvement of host cell factors in propilin maturation. At the nonpermissive temperature in a LepBts (leader peptidase B) host, propilin processing was inhibited. Furthermore, under these conditions, only full-length precursor was observed, suggesting that LepB is responsible for the removal of the entire propilin leader peptide. Using propilin processing as a measure of propilin insertion into the plasma membrane, we found that inhibition or depletion of SecA and SecY does not affect propilin maturation. Addition of a general membrane perturbant such as ethanol also had no effect. However, dissipation of the proton motive force did cause a marked inhibition of propilin processing, indicating that membrane insertion requires this energy source. We propose that propilin insertion in the plasma membrane proceeds independently of the SecA-SecY secretion machinery but requires the proton motive force. These results present a model whereby propilin insertion leads to processing by leader peptidase B to generate the 7-kDa peptide, which is then acetylated in the presence of TraX.
Adenosine Triphosphatases/metabolism; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins/metabolism; Cell Membrane/metabolism; Cloning, Molecular; Endopeptidases/metabolism; Escherichia coli Proteins; Ethanol/pharmacology; Fimbriae Proteins; Gene Expression; Membrane Proteins/metabolism; Membrane Transport Proteins; Protein Precursors/genetics; Protein Precursors/metabolism; Protein Processing, Post-Translational/drug effects; Proton-Motive Force; Serine Endopeptidases
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