Establishment of a reporter system for monitoring activation of the ER stress transducer ATF60

Abstract

ATF6 has two isoforms, ATF6a and ATF60, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional roles of ATF6a in response to ER stress have been well-studied, those of its paralogue ATF60 are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF60 activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF60. This system expresses a chimeric protein containing a synthetic transcription factor followed by the trans membrane domain and C-terminal luminal domain of ATF60. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF60. ? 2021 Elsevier Inc. All rights reserved.