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

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 Ca2+ 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 Ca2+-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.