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
Affiliations
Hiroshi Kadokura
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan; Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Kanagawa 226-8501, Japan; Corresponding author
Nanshi Harada
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan
Satoshi Yamaki
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan
Naoya Hirai
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan; Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Kanagawa 226-8501, Japan
Ryusuke Tsukuda
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan
Kota Azuma
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan
Yuta Amagai
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan; Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka 812-8582, Japan
Daisuke Nakamura
Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan
Kota Yanagitani
Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
Hideki Taguchi
Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Kanagawa 226-8501, Japan
Kenji Kohno
Institute for Research Initiatives, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; Graduate School of Science, University of Hyogo, Harima Science Garden City, Hyogo 678-1297, Japan
Kenji Inaba
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan; Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka 812-8582, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo 100-0004, Japan
Summary: 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.
