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

• Published in: iScience 2024-11, Vol.27 (11), p.111189, Article 111189
• Main Authors: Kadokura, Hiroshi, Harada, Nanshi, Yamaki, Satoshi, Hirai, Naoya, Tsukuda, Ryusuke, Azuma, Kota, Amagai, Yuta, Nakamura, Daisuke, Yanagitani, Kota, Taguchi, Hideki, Kohno, Kenji, Inaba, Kenji
• Format: Article
• Language: English
• Subjects: Bioengineering; Cell biology; Protein structure aspects
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2024.111189

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. [Display omitted] •We engineered firefly luciferase to detect protein biogenesis abnormalities at the ER•The reporter proteins exhibited disulfide-mediated misfolding under normal conditions•The reporter system identified defects in disulfide bond formation or ER localization•It detected a localization defect induced by an inhibitor of virus receptor synthesis Bioengineering; Cell biology; Protein structure aspects