A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag
Abstract
Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the GFP-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g., GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant RFP accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g., Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, S1, S2, 3, and 4.
Article and author information
Author details
Funding
Japan Science and Technology Agency (Exploratory Research for Advanced Technology (ERATO),JPMJER1702)
- Noboru Mizushima
Japan Society for the Promotion of Science (Transformative Research Areas (A),21H05256)
- Hayashi Yamamoto
Japan Society for the Promotion of Science (Specially Promoted Research,22H04919)
- Noboru Mizushima
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Li Yu, Tsinghua University, China
Version history
- Received: March 24, 2022
- Preprint posted: April 13, 2022 (view preprint)
- Accepted: August 8, 2022
- Accepted Manuscript published: August 8, 2022 (version 1)
- Version of Record published: August 17, 2022 (version 2)
Copyright
© 2022, Yim et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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