Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

Hiroshi Kadokura; Nanshi Harada; Satoshi Yamaki; Naoya Hirai; Ryusuke Tsukuda; Kota Azuma; Yuta Amagai; Daisuke Nakamura; Kota Yanagitani; Hideki Taguchi; Kenji Kohno; Kenji Inaba

doi:10.1016/j.isci.2024.111189

Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities

iScience. 2024 Oct 16;27(11):111189. doi: 10.1016/j.isci.2024.111189. eCollection 2024 Nov 15.

Authors

Hiroshi Kadokura^{1

2

3}, Nanshi Harada¹, Satoshi Yamaki¹, Naoya Hirai^{1

3}, Ryusuke Tsukuda¹, Kota Azuma¹, Yuta Amagai^{1

4}, Daisuke Nakamura², Kota Yanagitani^{2

5}, Hideki Taguchi³, Kenji Kohno^{2

6}, Kenji Inaba^{1

4

7}

Affiliations

¹ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan.
² Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
³ Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Kanagawa 226-8501, Japan.
⁴ Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka 812-8582, Japan.
⁵ Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
⁶ Graduate School of Science, University of Hyogo, Harima Science Garden City, Hyogo 678-1297, Japan.
⁷ Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo 100-0004, Japan.

Abstract

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.

Keywords: Bioengineering; Cell biology; Protein structure aspects.