Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

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 reported by large, non-natural tags that perturb 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 protein transport in bacteria. Under the conditions deployed, transport of a model pre-protein substrate (proSpy) occurs at 200 amino acids per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is best described by a series of large, ~30 amino acid, steps each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.