Delta psi stimulates membrane translocation of the C-terminal part of a signal sequence.
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.
van Dalen
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Department of Biochemistry of Membranes, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. a.vandalen@chem.uu.nl
Killian
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de Kruijff
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B
D000595:Amino Acid Sequence; D019286:Endopeptidase K; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D018272:Porins / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D055672:Static Electricity; D013499:Surface Properties
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D018272:Porins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011510:Proteolipids; C020485:proteoliposomes; C082922:PhoE protein, E coli; D012697:Serine Endopeptidases; D019286:Endopeptidase K; C026515:type I signal peptidase