Bacterial Sec Protein Transport is Rate-limited by Precursor Length: A Single Turnover Study

Abstract

An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.

Position-dependent Effects of Polylysine on Sec Protein Transport

“…Consequently, the implication of the SecA conformational cycling model is that the kinetics of translocation should be reasonably well described by a model consisting of a series of first-order reaction steps. Surprisingly, the transport kinetics observed with our recently developed real time fluorescence-based assay appear inconsistent with this stepwise translocation model (24).…”

“…Proteins-Wild type proOmpA-HisC was described previously (24). Lysine residues were introduced at the locations indicated in the figures via inverse PCR (44,45).…”

“…The coding region of all plasmid constructs was confirmed by DNA sequencing. Precursor proteins were purified as described earlier (24), with the exception that protein expression was initiated when the A 600 reached ϳ2. Fluorescent proteins were labeled with Atto565 maleimide (24).…”

“…A SecA "clamp" appears to be properly positioned to hold the preprotein at some step of the translocation process (22). Precursor length dependence studies support a model in which translocation through the pore is the slow step of transport (23,24). Consequently, the implication of the SecA conformational cycling model is that the kinetics of translocation should be reasonably well described by a model consisting of a series of first-order reaction steps.…”

“…Our recently developed fluorescence-based assay with 1 s time resolution (24) provided transport efficiencies and detailed kinetic profiles of the translocation process for numerous charge mutants of proOmpA. The effect of charge was clearly dependent on where in the mature domain they were introduced.…”