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

Research output: Contribution to journalArticle (Academic Journal)peer-review

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 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.
Original languageEnglish
Number of pages9
JournalProceedings of the National Academy of Sciences of the United States of America
DOIs
Publication statusPublished - 30 Nov 2020

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