Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities
Localization to the endoplasmic reticulum (ER) and subsequent disulfide bond formation are crucial processes governing the biogenesis of secretory pathway proteins in eukaryotes. Hence, comprehending the mechanisms underlying these processes is important. Here, we have engineered firefly luciferase (FLuc) as a tool to detect deficiencies in these processes within mammalian cells. To achieve this, we introduced multiple cysteine substitutions into FLuc and targeted it to the ER. The reporter exhibited FLuc activity in response to defects in protein localization or disulfide bond formation within the ER. Notably, this system exhibited outstanding sensitivity, reproducibility, and convenience in detecting abnormalities in these processes. We applied this system to observe a protein translocation defect induced by an inhibitor of HIV receptor biogenesis. Moreover, utilizing the system, we showed that modulating LMF1 levels dramatically impacted the ER's redox environment, confirming that LMF1 plays some critical role in the redox control of the ER.
Authors
- Naoya Hirai
- Kenji Kohno (ORCID: https://orcid.org/0000-0002-3503-6551)
- Daisuke Nakamura (ORCID: https://orcid.org/0000-0003-0102-7478)
- Kenji Inaba (ORCID: https://orcid.org/0000-0001-8229-0467)
- Kota Yanagitani (ORCID: https://orcid.org/0000-0002-5905-9950)
- Yuta Amagai (ORCID: https://orcid.org/0000-0002-2999-4622)
- Hideki Taguchi (ORCID: https://orcid.org/0000-0002-6612-9339)
- Kota Azuma
- Hiroshi Kadokura (ORCID: https://orcid.org/0000-0001-7308-7141)
- Noboru Harada (ORCID: https://orcid.org/0000-0002-6791-7097)
- Satoshi Yamaki
- Ryoichi Tsukuda
Institutions
- Kyushu University (JP)
- University of Hyogo (JP)
- Tohoku University (JP)
- Japan Agency for Medical Research and Development (JP)
- Innovative Research (United States) (US)
- Nara Institute of Science and Technology (JP)
- Hyogo University (JP)
- The University of Osaka (JP)
Publication Details
- Journal
- iScience
- Published
- 2024-10-16
- DOI
- https://doi.org/10.1016/j.isci.2024.111189
- Citations
- 1
- Primary Topic
- Viral Infectious Diseases and Gene Expression in Insects
- Type
- article
- Field-Weighted Citation Impact
- 0.23
Funders
- Japan Foundation for Applied Enzymology
- Japan Agency for Medical Research and Development
- Ohsumi Frontier Science Foundation
- Japan Society for the Promotion of Science
- RIKEN
- Core Research for Evolutional Science and Technology