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

ABSTRACT: The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity-a prerequisite for understanding how biological systems work-has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) 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 modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

SUBMITTER: Allen WJ

PROVIDER: S-EPMC7749344 | biostudies-literature |

REPOSITORIES: biostudies-literature

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