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ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucoseregulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane domain, is primarily associated with the perinuclear region. Upon Tg stress, the ATF6 protein level dropped initially but quickly recovered with the additional appearance of a faster-migrating form. This new form of ATF6 was recovered as soluble nuclear protein by biochemical fractionation, correlating with enhanced nuclear localization of ATF6 as revealed by immunofluorescence. Optimal ATF6 stimulation requires at least two copies of the ERSE and the integrity of the tripartite structure of the ERSE. Of primary importance is a functional NF-Y complex and a high-affinity NF-Y binding site that confers selectivity among different ERSEs for ATF6 inducibility. In addition, we showed that YY1 interacts with ATF6 and in Tg-treated cells can enhance ATF6 activity. The ERSE stimulatory activity of ATF6 exhibits properties distinct from those of human Ire1p, an upstream regulator of the mammalian unfolded protein response. The requirement for a high-affinity NF-Y site for ATF6 but not human Ire1p activity suggests that they stimulate the ERSE through diverse pathways.

ATF6 is a key transcriptional activator of the unfolded protein response (UPR), which allows mammalian cells to maintain cellular homeostasis when they are subjected to a variety of environmental and physiological stresses that target the endoplasmic reticulum (ER). ATF6, a 90-kDa ER transmembrane protein, contains three evolutionarily conserved N-linked glycosylation sites within its carboxyl luminal domain. Although it is well established that p90ATF6 activation requires transit from the ER to the Golgi, where it is cleaved by the S1P/S2P protease system to generate a nuclear form p60ATF6 that acts as a transcriptional activator, the functional significance of p90ATF6 N-linked glycosylation is unknown. Here we show that ER Ca 2؉ depletion stress, a triggering mechanism for the UPR, induces the formation of ATF6(f), which represents de novo partial glycosylation of newly synthesized p90ATF6. By mutating a single amino acid within the N-linked glycosylation site closest to the carboxyl terminus of p90ATF6, we recreated ATF6(f). This mutation sharply reduces p90ATF6 association with calreticulin, a major Ca 2؉ -binding chaperone for N-glycoprotein. We further determined that ATF6(f) exhibits a faster rate of constitutive transport to the Golgi, resulting in a higher level of p60ATF6 in the nucleus and stronger transactivating activity in the absence of ER stress. Additional analysis of p90ATF6 mutants targeting single or multiple N-glycosylation sites also showed higher constitutive transactivating activity than wild type ATF6. Because accumulation of underglycosylated proteins in the ER is a potent inducer for the UPR, these studies uncover a novel mechanism whereby the glycosylation status of p90ATF6 can serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the UPR.The discovery that ATF6 is a key transcriptional activator of endoplasmic reticulum (ER) 1 -resident molecular chaperones and folding enzymes in the unfolded protein response (UPR) has revealed novel molecular mechanisms employed by mammalian cells to respond to stresses that perturb ER homeostasis. ATF6, a 670-amino acid glycoprotein with the electrophoretic mobility of a 90-kDa protein (p90ATF6), is constitutively expressed in a variety of mammalian cells (1). It contains a single transmembrane domain with 272 amino acids oriented in the ER lumen. ER stress induces proteolysis of the membrane-bound p90ATF6, releasing the soluble amino portion of p60ATF6, which relocates to the nucleus and activates the transcription of a wide variety of ER stress-inducible promoters, of which Grp78/BiP is the most well characterized (2-4). Conservation of protease cleavage sites led to the discovery that Golgi-localized S1P/S2P proteases that process sterol response element binding proteins in response to cholesterol deprivation also process ATF6 in response to ER stress (4). Specific luminal domains of p90ATF6 have been mapped which are required for translocation to the Golgi (5). It was further determined that GRP78 retains p90ATF6 in the ...

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