Underglycosylation of ATF6 as a Novel Sensing Mechanism for Activation of the Unfolded Protein Response

Abstract

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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