Abstract
The membrane-bound transcription factor ATF6α is activated by proteolysis during endoplasmic reticulum (ER) stress. ATF6α target genes encode foldases, chaperones, and lipid biosynthesis enzymes that increase protein-folding capacity in response to demand. The off-state of ATF6α is maintained by its spatial separation in the ER from Golgi-resident proteases that activate it. ER stress induces trafficking of ATF6α. We discovered Ceapins, a class of pyrazole amides, as selective inhibitors of ATF6α signaling that do not inhibit the Golgi proteases or other UPR branches. We show that Ceapins block ATF6α signaling by trapping it in ER-resident foci that are excluded from ER exit sites. Removing the requirement for trafficking by pharmacological elimination of the spatial separation of the ER and Golgi apparatus restored cleavage of ATF6α in the presence of Ceapins. Washout of Ceapins resensitized ATF6α to ER stress. These results suggest that trafficking of ATF6α is regulated by its oligomeric state.
Highlights
Activating transcription factor six alpha (ATF6a) is a type-II transmembrane protein localized in the endoplasmic reticulum (ER) where, with its close homolog ATF6b, it functions as an ER stress sensor in one of the three principal branches of the unfolded protein response (UPR) (Haze et al, 1999; Gardner et al, 2013)
We describe the identification of Ceapins, a class of pyrazole amides that inhibit selectively the processing of ATF6a by S1P and S2P in response to ER stress but not the other UPR sensors, including – surprisingly – ATF6b, or SREBP
We followed nuclear translocation of GFPATF6a-N, the proteolytic fragment resulting from GFP- ATF6a cleavage in response to ER stress
Summary
Activating transcription factor six alpha (ATF6a) is a type-II transmembrane protein localized in the endoplasmic reticulum (ER) where, with its close homolog ATF6b, it functions as an ER stress sensor in one of the three principal branches of the unfolded protein response (UPR) (Haze et al, 1999; Gardner et al, 2013). ATF6a target genes are exclusively cytoprotective, functioning to increase the folding capacity of the ER and restore ER homeostasis (Adachi et al, 2008; Wu et al, 2007). When demand exceeds the folding capacity of the ER, ATF6a is transported from the ER to the Golgi apparatus, where sequential cleavage by two Golgi-resident proteases – site-1 and site-2 proteases (S1P and S2P), respectively - releases its N-terminal domain (ATF6a-N) from the membrane as a functional b-Zip transcription factor. ATF6a-N is imported into the nucleus (nuclear translocation) where it activates transcription of its target genes (Ye et al, 2000). The mechanism that retains ATF6a in the ER and releases it to allow transport to the Golgi apparatus is unknown. Deciphering the mechanism of ATF6a’s regulated trafficking is essential to understanding how proteostasis is maintained in the ER
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