Δψ Stimulates membrane translocation of the C-terminal part of a signal sequence

Abstract

For several proteins in Escherichia coli it has been shown that the protonmotive force (pmf) dependence of translocation can be varied with the signal sequence composition, suggesting an effect of the pmf on the signal sequence. To test this possibility, we analyzed the effect of the membrane potential on translocation of the signal sequence. For this purpose, a precursor peptide was used (SP+7), corresponding to the signal sequence of PhoE with the first seven amino acids of the mature part that can be processed by purified leader peptidase. Translocation was studied in pure lipid vesicles containing leader peptidase, with its active site inside the vesicles. In the presence of a positive inside Δψ, the amount of processing of SP+7 was significantly higher than without a Δψ, indicating that the translocation of the cleavage region is stimulated by Δψ. Replacement of the helix-breaking glycine residue at position -10 in the signal sequence for a leucine abolished the effect of Δψ on the translocation of the cleavage region. It is concluded that Δψ directly acts on the wild type signal sequence by stimulating the translocation of its C terminus. We propose that Δψ acts on the signal sequence by stretching it into a transmembrane orientation.