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

Abstract

Abbreviations:AA -antimycin A; IMM -inner mitochondrial membrane; mt-11S -mitochondrial-targeted 11S; H6 11S -Nterminal 6xHis tagged version of 11S; OMM -outer mitochondrial membrane; OVA -oligomycin, valinomycin and antimycin A (full depolarisation cocktail); MTS -mitochondrial targeting sequence, NanoBiT -NanoLuc Binary Technology; PAM -presequence-translocase-associated import-motor; pep86high-affinity version of NanoBiT small fragment; pep114 -small fragment of NanoBiT; PMF -proton motive force; TIM -translocase of the inner membrane; TOM -translocase of the outer membrane; 11Slarge fragment of the NanoBiT; Δ Ψ -membrane potential. 2 ABSTRACTProtein translocation is a fundamental process in biology. Major gaps in our understanding of this process arises 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 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 rigour unattainable through classical methods.

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

“…Using NanoLuc to Dissect Translocation Kinetics. To interrogate the kinetics of protein transport in sufficient detail to reveal mechanistic information, we used the recently developed NanoLuc system (16). In essence, NanoLuc luciferase missing a single β-strand (11S) is encapsulated within proteoliposomes (PLs) incorporating the Sec machinery, while a high-affinity version of the missing β-strand (Pep86) is fused to a translocation substrate.…”

“…Very precise measurements of protein transport are required to distinguish between these types of mechanisms, such as those produced by the recently published NanoLuc transport assay (16). Here, we extend the use of the NanoLuc assay to reveal the elementary steps of the ATP-driven protein transport mechanism, using the model preprotein prospheroplast protein Y (pSpy).…”

The SecA motor generates mechanical force during protein translocation

“…To analyze translocation kinetics at high time resolution, we utilized a recently described assay 33 that continuously follows the reaction progress in real-time through light emission by the NanoLuc luciferase. NanoLuc can be split into two asymmetric fragments p86 (11 amino acids) and 11S (159 amino acids), neither of which has significant enzymatic activity.…”

“…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) 33 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. Expression was induced with a final concentration of 0.2% arabinose at 37 °C when the OD600 reached 0.4–0.6.…”

A NanoLuc luciferase‐based assay enabling the real‐time analysis of protein secretion and injection by bacterial type III secretion systems

“…Since the NanoLuc reaction follows a Michaelis–Menten kinetics (Hall et al, ), use of this assay does even allow estimating the actual concentration of secreted or injected proteins. The herein described set‐up has been successfully used for assessing the function of the Yersinia T3SS (Lindner, Milne‐Davies, Langenfeld, & Diepold, ) and it was shown that a similar set‐up also works for monitoring secretion through the Sec‐translocon (Pereira et al, ). Thus, NLuc fusions have a high potential to also enable the functional analysis of other bacterial secretion systems in the future.…”