Related Experiment Video
Updated: Dec 27, 2025
Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay
Published on: September 10, 2020
Rapid changes in the ATG5-ATG16L1 complex following nutrient deprivation measured using NanoLuc Binary Technology
Emily Crowley1, Euphemia Leung2, Jóhannes Reynisson1
1School of Pharmacy and Bioengineering, Guy Hilton Research Centre, Keele University, Stoke-on-Trent, UK.
Abstract:
Autophagy plays a role in several human diseases, but each of the current methods to measure autophagy has significant drawbacks. ATG5 and ATG16L1 are regulators necessary for autophagy; therefore, drugs that inhibit the interaction of these proteins may be therapeutically useful. To evaluate the interaction of ATG5 and ATG16L1 in cells, their cDNAs were fused to the coding sequences of SmBIT and LgBIT, two components of NanoLuc luciferase. This generated a luminescent signal when SmBIT and LgBIT interacted to form a functional luciferase as a result of their colocalization that was brought about by the binding of ATG5 and ATG16L1. The assay measures the interaction in real time and can be used in microplate format to allow for multiple experimental conditions to be assessed. The interaction of ATG5 and ATG16L1 is not significantly altered by inhibition of lysosomal function, or inhibitors of Ulk1, Vps34 or mTORC1. However, there was a constitutive interaction of ATG5 and ATG16L1 and luminescence was stimulated within 3 min, by up to 500%, when the cells were deprived of nutrients. When the nutrients are returned, the complex returns to its basal status equally rapidly. Sphingosine-1-phosphate and CYM-5541 partially repressed the effects of nutrient starvation. Furthermore, we identified a small-molecule inhibitor that interferes with the interaction of ATG5 and ATG16L1 in cells. This assay provides a novel tool for researchers to measure autophagy and can be potentially applied to many cell types. DATABASE: Replicate data are available in Figshare database https://doi.org/10.6084/m9.figshare.11798946.
Insights
Researchers developed a novel luminescence assay to measure autophagy by tracking the interaction of ATG5 and ATG16L1 proteins. This assay enables real-time monitoring and identifies a new inhibitor for autophagy regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Autophagy is crucial in human diseases, but current measurement methods have limitations.
- ATG5 and ATG16L1 are essential regulators of autophagy, making their interaction a therapeutic target.
Purpose of the Study:
- To develop a novel, real-time luminescence assay for measuring ATG5 and ATG16L1 interaction in cells.
- To identify modulators of ATG5 and ATG16L1 interaction and potential therapeutic targets for autophagy-related diseases.
Main Methods:
- Constructed a luminescence-based assay by fusing ATG5 and ATG16L1 cDNAs to NanoLuc luciferase fragments (SmBIT and LgBIT).
- Measured luminescence in real-time to quantify ATG5 and ATG16L1 complex formation in response to cellular conditions.
- Utilized microplate format for high-throughput screening of autophagy modulators.
Main Results:
- The assay demonstrated a constitutive interaction of ATG5 and ATG16L1, with luminescence increasing up to 500% upon nutrient deprivation.
- The interaction rapidly returned to basal levels upon nutrient repletion.
- Identified a small-molecule inhibitor that disrupts ATG5 and ATG16L1 interaction, with partial repression observed by sphingosine-1-phosphate and CYM-5541.
Conclusions:
- The developed luminescence assay provides a sensitive and dynamic tool for studying autophagy.
- This assay can be applied to various cell types for drug screening and understanding autophagy regulation.
- The findings offer new insights into the regulation of ATG5 and ATG16L1 interaction and its potential as a therapeutic target.
