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

Abstract

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.

Rate-limiting transport of positive charges through the Sec-machinery is integral to the mechanism of protein transport

“…Transport reaction components were produced as described previously 27 . To make inverted membrane vesicles (IMVs) at different pHs, a standard protocol was followed 35 coli strain BL21(DE3), which produces a PMF in the presence of ATP, or HB1(DE3), which lack ATP synthase, and therefore does not 43 .…”

“…While the classical import assay above is useful in that it is compatible with low pH, it does not provide much detail on the kinetics of protein import. For subsequent import assays we therefore switched to our newly developed NanoLuc import assay 27 (Fig. 2b).…”

“…For the transport of the proSpy Lys/Arg series (Fig. 3a), assays were conducted at pH 8.0 in a 96-well plate with a plate reader (Synergy Neo2, Biotek), as described 27 at the manufacturer's recommended concentration (1/100). From the raw traces, the time required for transport to reach 50% completion was determined (t50%), and its reciprocal used as a measure of transport rate (kapp; see Fig.…”

“…The model mitochondrial import substrate B2 #$%&'( ))* -Pep86 was expressed and purified as described previously 27…”

The SecA motor generates mechanical force during protein translocation

“…The codon optimized 11S sequence was obtained as a synthetic DNA fragment (IDT DNA) and cloned into a His-SUMO backbone (Addgene Plasmid #37507) for expression. The coding sequence for the p86 "dark" peptide (VSGWALFKKIS) 31 was inserted into a plasmid encoding glutathione S transferase (GST) to generate a C-terminal fusion in the same backbone and termed "GST-dark." For expression, plasmids were transformed into BL21(DE3) cells.…”