Requirement for conformational flexibility in the signal sequence of precursor protein.
According to the "unlooping" model (de Vrije, T., Batenburg, A. M., Killian, J. A., and de Kruijff, B. (1990) Mol. Microbiol. 4, 143-150), proposed to explain how signal sequences serve to target proteins into the secretory pathway, the initial interaction of the signal sequence with the membrane in a helix-turn-helix conformation (spanning half of the bilayer) plays an important role in the initiation of the translocation reaction. To test this model we have introduced 2 cysteines (at positions -5 and -19) in the signal sequence of the Escherichia coli outer membrane protein PhoE. The mutations did not influence the translocation of precursor PhoE in vivo or in vitro. The 2 cysteines were oxidized to form a disulfide bridge. In vitro translocation of the looped precursor into inner membrane vesicles was disturbed. The looped precursor competed with translocation of wild type precursor PhoE, and looped precursor that was first bound to inner membrane vesicles could be translocated after the addition of dithiothreitol. Apparently, the mutant precursor with a disulfide bridge in the signal sequence is arrested as a very early intermediate in the translocation process. All of these results are consistent with the proposed unlooping model and show that, besides the primary structure characteristics of a signal sequence, conformational flexibility is needed to initiate the translocation reaction.
Nouwen
N
N
Institute of Biomembranes, Utrecht University, The Netherlands.
Tommassen
J
J
de Kruijff
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D001692:Biological Transport; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004220:Disulfides; D029968:Escherichia coli Proteins; D018272:Porins; D011498:Protein Precursors; D021382:Protein Sorting Signals; C082922:PhoE protein, E coli; D003545:Cysteine