ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes.

Abstract

The precursor protein proOmpA can translocate across purified Escherichia coli inner membrane vesicles in the absence of any other soluble proteins. ProOmpA, purified 2000‐fold in the presence of 8 M urea, is competent for translocation following rapid renaturation via dilution. ATP, the transmembrane electrochemical potential, and functional secY protein are essential for the translocation of proOmpA renatured by dilution. The kinetics of its translocation and the level of translocation at each concentration of ATP are indistinguishable from that of proOmpA renatured by dialysis with trigger factor. After dilution, the proOmpA rapidly loses its competence for membrane assembly. However, this competence is stabilized by trigger factor. Assembly‐competent proOmpA is in a protease‐sensitive conformation, whereas proOmpA which has lost this competence is more resistant to degradation. This suggests that the primary role for trigger factor in in vitro protein translocation is to maintain precursor proteins in a translocation‐competent conformation. We propose that a properly folded precursor protein and ATP are the only soluble components which are essential for bacterial protein translocation.

Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules

“…OmpT was expressed from plasmid pND9 in strain SF100 and expressed under its own temperature sensitive promoter (29). The proOmpA cysteine mutant S245C was constructed with the QuickChange site-directed mutagenesis kit (Stratagene) using pET2345 containing the cysteineless proOmpA as a template (30). Primers used introduced in addition a silent MluI cutting site for cloning purposes: S245C forward primer, ccgaccgcat cggttgtgac gcgtacaacc agggtctg; S245C reverse primer, cagaccctgg ttgtacgcgt cacaaccgat gcggtcgg.…”

“…The introduced mutations were confirmed by sequencing. ProOmpA(S245C) was purified as described previously (30) and further referred to as proOmpA.…”

Kinetic Analysis of the Translocation of Fluorescent Precursor Proteins into Escherichia coli Membrane Vesicles

“…pET502 was used as template to construct the double cysteine mutant proOmpA (C302S,D291C,proOmpA-Cys-290,Cys-291) (pET2349). Purification and Labeling of proOmpA-ProOmpA was purified as described previously (13). Prior to labeling, the proOmpA (3.5 mg/ml in 8 M urea, 50 mM Tris-HCl, pH 7.0) was reduced with 1 mM tris-(2-carboxyethyl)phosphine and incubated with 2 mM fluorescent maleimide at room temperature for 30 min.…”

Identification of the prolyl isomerase domain of Escherichia coli trigger factor

“…E. coli trigger factor was originally identified by its activity to stimulate membrane translocation of the precursor of the outer membrane protein A (proOmpA) in vitro. Based on its stoichiometric binding to this substrate it was proposed to act as a secretion specific chaperone [3,4]. However, subsequent investigations showed that cells depleted of trigger factor had no secretion defect for proOmpA [S].…”