MEMBRANE INSERTION OF THE F-PILIN SUBUNIT IS SEC-INDEPENDENT BUT REQUIRES LEADER PEPTIDASE-B AND THE PROTON MOTIVE FORCE

F pilin is the subunit required for the assembly of conjugative pill o n the cell surface of Escherichia coli carrying the F plasmid, Maturat ion of the F-pilin precursor, propilin, involves three F plasmid trans fer products: TraA, the propilin precursor; TraQ, which promotes effic ient 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 e xperiments to determine the involvement of host cell factors in propil in maturation, At the nonpermissive temperature in a LepB(ts) (leader peptidase B) host, propilin processing was inhibited, Furthermore, und er these conditions, only full-length precursor was observed, suggesti ng that LepB is responsible for the removal of the entire propilin lea der peptide, Using propilin processing as a measure of propilin insert ion into the plasma membrane, we found that inhibition or depletion of SecA and SecY does not affect propilin maturation, Addition of a gene ral membrane perturbant such as ethanol also had no effect. However, d issipation 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 membr ane proceeds independently of the SecA-SecY secretion machinery but re quires the proton motive force, These results present a model whereby propilin insertion leads to processing by leader peptidase B to genera te the 7-kDa peptide, which is then acetylated in the presence of TraX .