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bioRxiv · 10.1101/2020.05.08.084160

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

Allen, W. J.·Watkins, D. W.·Dillingham, M. S.·Collinson, I.
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Abstract

The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity - a prerequisite for understanding how biological systems works - has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, [~]30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient - in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

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Allen, W. J., Watkins, D. W., Dillingham, M. S., Collinson, I.. 2020-05-09. Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover. https://doi.org/10.1101/2020.05.08.084160

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