title: Luminal domain of ATF6 alone is sufficient for sensing endoplasmic reticulum stress and subsequent transport to the Golgi apparatus.
abstract: The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol- or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Sato
authors.forename: Satomi
authors.initials: S
authors.lastname: Nadanaka
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Okada
authors.forename: Katsuya
authors.initials: K
authors.lastname: Okawa
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Mori
mesh_terms: D051702:Activating Transcription Factor 6 / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D004220:Disulfides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary
chemical_list: D051702:Activating Transcription Factor 6; D004220:Disulfides; D006360:Heat-Shock Proteins