Recent Publications (Original)
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag (Yim et al., eLife)
2022.08.16 Recent Publications (Original)
Willa Wen-You Yim , Hayashi Yamamoto & Noboru Mizushima
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag
Elife. 2022 Aug 8;11:e78923. doi: 10.7554/eLife.78923. Online ahead of print.
Based on our finding that HaloTag becomes resistant to proteolysis upon ligand binding, we developed the HaloTag processing assay. Pulse-labeled HaloTag reporters are processed in lysosomes, releasing proteolysis-resistant labeled Halo that persist in lysosomes. Free labeled Halo amount = autophagic flux.
This method is
- Lysosomal inhibitor-free
- External control-free
- Adaptable to monitor most autophagy pathways or autophagic degradation of a protein of interest
In our study, we demonstrate the HaloTag processing assay for
- General (LC3-dependent) autophagy – Halo-LC3, Halo-GFP-LC3, Halo-GFP-LC3-RFP
- Selective autophagy of ER or mitochondria – Halo-GFP-KDEL, pSu9-Halo-GFP
- Bulk non-selective (LC3-independent) autophagy – Halo-GFP
The plasmids can be found on Addgene (https://www.addgene.org/browse/article/28225176/).