A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring p...

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Bibliographic Details
Published in:Journal of molecular biology 2019-04, Vol.431 (8), p.1689-1699
Main Authors: Pereira, Gonçalo C., Allen, William J., Watkins, Daniel W., Buddrus, Lisa, Noone, Dylan, Liu, Xia, Richardson, Andrew P., Chacinska, Agnieszka, Collinson, Ian
Format: Article
Language:English
Subjects:
bacterial Sec system
Escherichia coli - metabolism
Escherichia coli Infections - microbiology
Escherichia coli Proteins - metabolism
Humans
live assay
Luciferases - metabolism
Luminescent Agents - metabolism
Luminescent Measurements - methods
Mitochondrial Membranes - metabolism
mitochondrial protein import
NanoLuc
protein translocation
Protein Transport
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - metabolism
SEC Translocation Channels - metabolism
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Summary:Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods. [Display omitted] •Conventional methods for monitoring protein translocation are laborious and discontinuous and lack kinetic detail.•A split NanoLuc system was adapted for real-time monitoring of protein translocation through the bacterial Sec system and the mitochondrial Tim23 complex.•The new assay reduces the timescale of data acquisition from days to minutes.•It produces high-quality results suitable for kinetic fitting and model derivation.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2019.03.007