Regulating the trafficking of the unfolded protein response (UPR) reporter ATF6

Tungkum, Wanida (2023) Regulating the trafficking of the unfolded protein response (UPR) reporter ATF6. PhD thesis, University of Glasgow.

Abstract

The activating transcription factor 6 (ATF6) is one of the three membrane proteins, which responds to misfolded protein induced stress, in the endoplasmic reticulum (ER). During ER stress, ATF6 dissociates from BiP, an ER chaperone, then transports to the Golgi apparatus where ATF6 undergoes proteolytic processing by site 1 protease (S1P) and site 2 protease (S2P). Up until now, the dissociation of BiP from ATF6 is reported to be the key factor that regulates the trafficking of ATF6 from the ER to Golgi in the unfolded protein response (UPR). However, our study suggests that the ATF6-BiP complex disassembly is not the only factor which controls ATF6 migration when cells get induced ER stress by standard inducers like dithiothreitol (DTT) and thapsigargin (TG). To identify the ATF6 protein complex, the stable cell-lines of the non-trafficking mutants ATF6α, D564G and Y567N, were created and compared with the wild type ATF6α. From our studies, the ATF6α mutants show specific characteristics, distinctively from the wild type ATF6α, which are likely to affect the ability of ATF6 to bind with other proteins and to form disulfides. The results of mass spectrometry indicate numerous proteins which seem to be interacted with ATF6 along with BiP such as 14 3-3E, Neudesin, TFG and ERdj3. Unfortunately, we can only properly investigate ERdj3. We investigated the interaction of ERdj3 protein with ATF6 by immunoisolation and western blot; the results show that ERdj3 interacts with ATF6. Subsequently, the lipotoxic stress inducers, dihydrosphingosine (DHS) and dihydroceramide (DHC), were used to induce ER stress and ATF6 pathway since it was reported that lipotoxic stress upregulates ATF6 differently compared with the mechanism of proteotoxic stress. However, the results from our study with the wild type ATF6α cell-line show that DHS and DHC did not upregulate the ATF6 stress response as previously reported. Taken together our results define the interactome of ERlocalised ATF6α and suggest a role for ERdj3 during the ER stress response.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Life Sciences > Life Sciences Biomolecular Science
• Supervisor's Name: Bulleid, Professor Neil
• Date of Award: 2023
• Depositing User: Theses Team
• Unique ID: glathesis:2023-83743
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Jul 2023 09:05
• Last Modified: 27 Jul 2023 09:08
• Thesis DOI: 10.5525/gla.thesis.83743
• URI: https://theses.gla.ac.uk/id/eprint/83743