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

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.