Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 50, Pages 31808-31816Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2010906117
Keywords
protein secretion; molecular motor; NanoLuc; SecYEG; SecA
Categories
Funding
- Biotechnology and Biological Sciences Research Council [BB/S008349/1, BB/N015126/1]
- Wellcome Trust [104632]
- BBSRC [BB/S008349/1, BB/N015126/1] Funding Source: UKRI
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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 (similar to 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.
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