Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form.
Pro-OmpA that is synthesized in vitro can assemble into bacterial inner membrane vesicles in the presence of ATP and NADH. We have purified pro-OmpA to determine which additional soluble proteins are necessary for its membrane assembly. [35S]Pro-OmpA was bound to Sepharose-linked antibody to OmpA, then eluted with 8 M urea and chromatographed on an anion-exchange resin in 8 M urea. This pro-OmpA is purified 2000-fold and is radiochemically pure. After dialysis, it is soluble but incompetent for membrane assembly. Addition of an Escherichia coli cytoplasmic fraction (S100) to the assembly reaction does not allow translocation. However, when S100 is added to pro-OmpA prior to dialysis, full assembly competence is restored, suggesting that a soluble factor, termed "trigger factor," triggers the folding of pro-OmpA into an assembly-competent form as the urea is removed. We noted that, prior to the last purification step, the immunoaffinity-purified pro-OmpA was partially competent for membrane assembly without addition of trigger factor. To test whether trigger factor had bound to the antibody column by means of its association with pro-OmpA, the crude pro-OmpA was acid-denatured prior to immunoadsorption. In this experiment, the trigger factor did not bind to the anti-OmpA column, and S100 was required for renaturation of this [35S]pro-OmpA. As suggested by this experiment, the crude [35S]pro-OmpA was in a complex with other proteins. Sedimentation velocity studies showed that the trigger factor has an apparent molecular weight of approximately 60,000. We propose that it is required for translocation-competent folding of pro-OmpA and other precursor proteins.
Crooke
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E
Wickner
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002474:Cell-Free System; D004926:Escherichia coli; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D012995:Solubility
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D009243:NAD; D000255:Adenosine Triphosphate