A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6

Okada T, Haze K, Nadanaka S, Yoshida H, Seidah NG, Hirano Y, Sato R, Negishi M and Mori K
J Biol Chem (2003) 278(33): 31024-31032
Cells cope with unfolded proteins accumulated in the endoplasmic reticulum (ER) primarily by transient attenuation of translation and by transcriptional induction of genes encoding ER-resident molecular chaperons and folding enzymes. This process is collectively termed as the unfolded protein response (UPR). ATF6 is an ER stress-regulated transmembrane transcription factor that activates the transcription of ER molecules. ATF6 is cleaved sequentially by the Site-1 protease (S1P) located in the Golgi apparatus. To gain further insights in the role of S1P in ATF6 translocation and cleavage HeLa cells were nucleofected with a series of plasmids encoding wild-type or mutant version of S1P.
Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6alpha and ATF6beta), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6alpha and ATF6beta, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6alpha and ATF6beta by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.