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Mol Cells. 2019 Nov 30;42(11):783-793. doi: 10.14348/molcells.2019.0104.

Development of a Reporter System Monitoring Regulated Intramembrane Proteolysis of the Transmembrane bZIP Transcription Factor ATF6α.

Author information

1
Department of Biochemistry & Health Sciences, Changwon National University, Changwon 51140, Korea.
2
Degenerative Diseases Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
3
School of Biological Sciences, University of Ulsan, Ulsan 44610, Korea.

Abstract

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of ATF6α by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human ATF6α N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-hATF6α deletion variant was cleaved to liberate active transcription activator encompassing GV-hATF6α fragment which could translocate into the nucleus. The translocated GV-hATF6α fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-hATF6α(333) represents an innovative tool to investigate regulated intramembrane proteolysis of ATF6α. It can substitute active pATF6(N) binding motif-based reporter cell lines.

KEYWORDS:

ER stress; GAL4 binding site; activating transcription factor 6; luciferase; regulated intramembrane proteolysis; reporter cell line; unfolded protein response

PMID:
31707777
DOI:
10.14348/molcells.2019.0104
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