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 Delta psi, the amount of processing of SP+7 was significantly higher than without a Delta psi, indicating that the translocation of the cleavage region is stimulated by Delta psi. Replacement of the helix-breaking glycine residue at position -10 in the signal sequence for a leucine abolished the effect of Delta psi on the translocation of the cleavage region. It is concluded that Delta psi directly acts on the wild type signal sequence by stimulating the translocation of its C terminus. We propose that Delta psi acts on the signal sequence by stretching it into a transmembrane orientation.
Highlights
Proteins destined for export out of the cell are synthesized as precursor proteins with an N-terminal extension, the signal sequence
Strategy—To investigate the effect of the pmf on the translocation of the signal sequence, a precursor peptide was used, corresponding to the signal sequence of PhoE with the first seven amino acids of the mature part (SPϩ7). In this peptide the cleavage region for leader peptidase (Lep) is intact. Processing of this precursor peptide in the lumen of reconstitution vesicles by Lep can in principle be used as a tool for translocation of the C terminus of the signal sequence
Whether the pmf directly acts on the signal sequence remained unclear, because effects via components of the secretion machinery could not be excluded
Summary
Proteins destined for export out of the cell are synthesized as precursor proteins with an N-terminal extension, the signal sequence. We demonstrate that the translocation of the cleavage region of the signal sequence across the bilayer is largely stimulated in the presence of ⌬ This stimulating effect was not observed with an analogous precursor peptide in which the GlyϪ10 was replaced by a Leu residue. This demonstrates that ⌬ can directly and act on the signal sequence and suggests that ⌬ affects the stretching of the signal sequence into a transmembrane conformation and thereby facilitates initiation of translocation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
