Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Development of a Reporter System Monitoring Regulated Intramembrane Proteolysis of the Transmembrane bZIP Transcription Factor ATF6α

  • Kim, Jin-Ik (Department of Biochemistry & Health Sciences, Changwon National University) ;
  • Kaufman, Randal J. (Degenerative Diseases Program, Sanford Burnham Prebys Medical Discovery Institute) ;
  • Back, Sung Hoon (School of Biological Sciences, University of Ulsan) ;
  • Moon, Ja-Young (Department of Biochemistry & Health Sciences, Changwon National University)
  • Received : 2019.05.22
  • Accepted : 2019.09.30
  • Published : 2019.11.30
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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 $ATF6{\alpha}$ 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 $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ 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-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

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References

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