title: Analysis of ATF6 activation in Site-2 protease-deficient Chinese hamster ovary cells.
abstract: Mammalian transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). It is activated when unfolded proteins are accumulated in the ER under ER stress through a process called regulated intramembrane proteolysis (Rip), in which ATF6 is transported from the ER to the Golgi apparatus where it undergoes sequential cleavage by Site-1 and Site-2 proteases. The cytosolic transcription factor domain of ATF6 liberated from the Golgi membrane enters the nucleus where it activates transcription of ER-localized molecular chaperones and folding enzymes, leading to the maintenance of the homeostasis of the ER. Here, we analyzed M19 cells, a mutant of Chinese hamster ovary cells deficient in Site-2 protease. It was previously shown that M19 cells are defective in the induction of mRNA encoding the major ER chaperone BiP. In M19 cells, ATF6 was not converted from the membrane-bound precursor form to the cleaved and nuclear form as expected. Moreover, some of the ATF6 was constitutively relocated to the Golgi apparatus, where it was cleaved by Site-1 protease, and remained associated with the Golgi apparatus, indicating that the ER of M19 cells was constitutively stressed. Consistent with this notion, the two other ER stress response mediators, IRE1 and PERK, were also constitutively activated in M19 cells. M19 cells showed inefficient secretion of a model protein. These results suggest that Rip-mediated activation of ATF6 is important for the homeostasis of the ER in not only ER-stressed but also unstressed cells.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Nadanaka
authors.forename: Hiderou
authors.initials: H
authors.lastname: Yoshida
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Sato
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Mori
mesh_terms: D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004789:Enzyme Activation; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: D051702:Activating Transcription Factor 6; D004268:DNA-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D008666:Metalloendopeptidases; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D043484:Proprotein Convertases; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D012697:Serine Endopeptidases; D014157:Transcription Factors; D019892:eIF-2 Kinase; C486556:Ern2 protein, mouse; C115575:membrane-bound transcription factor peptidase, site 1