CELL STRUCTURE AND FUNCTION 33: 75–89 (2008) © 2008 by Japan Society for Cell Biology ATF6 Is a Transcription Factor Specializing in the Regulation of Quality Control Proteins in the Endoplasmic Reticulum Yusuke Adachi1, Keisuke Yamamoto1, Tetsuya Okada1, Hiderou Yoshida1, Akihiro Harada2, and Kazutoshi Mori1∗ 1Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, 2Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan ABSTRACT. Eukaryotic cells cope with endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR), a coordinated system of transcriptional and translational controls, which ensures the integrity of synthesized proteins. Mammalian cells express three UPR transducers in the ER, namely IRE1, PERK and ATF6. The IRE1 pathway, which is conserved from yeast to humans, mediates transcriptional induction of not only ER quality control proteins (molecular chaperones, folding enzymes and components of ER-associated degradation) but also proteins working at various stages of secretion. The PERK pathway, conserved in metazoan cells, is responsible for translational control and also participates in transcriptional control in mammals. ATF6 is an ER- membrane-bound transcription factor activated by ER stress-induced proteolysis which consists of two closely related factors, ATF6α and ATF6β, in mammals. ATF6α but not ATF6β plays an important role in transcriptional control. In this study, we performed a genome-wide search for ATF6α-target genes in mice. Only 30 of the 14,729 analyzable genes were identified as specific targets, of which 40% were ER quality control proteins, 20% were ER proteins, while the rest had miscellaneous functions. The negative effects of the absence of PERK on transcriptional induction of ER quality control proteins could be explained by its inhibitory effect on ATF6α activation. Further, proteins involved in transport from the ER are not regulated by ATF6α, and transport of folded cargo molecules from the ER was not affected by the absence of ATF6α. Based on these results, we propose that ATF6 is a transcription factor specialized in the regulation of ER quality control proteins. Key words: endoplasmic reticulum/protein folding/protein degradation/transcription factor/microarray analysis Introduction cated back to the cytosol, where they are ubiquitinated and degraded by the proteasome through a process termed ER- Newly synthesized secretory and transmembrane proteins associated degradation (ERAD). These two mechanisms, are translocated into the endoplasmic reticulum (ER), which productive folding and ERAD, ensure the quality of pro- contains a number of molecular chaperones and folding teins that pass through the ER and allow only correctly enzymes (collectively termed ER chaperones hereafter) and folded molecules to move along the secretory pathway provides an optimal environment for the productive folding (Bukau et al., 2006). However, the ER quality control sys- of these proteins. Proteins remaining unfolded or misfolded tem is compromised under a variety of conditions, collec- even after the assistance of ER chaperones are retrotranslo- tively termed ER stress, resulting in the accumulation of unfolded proteins in the ER. Essentially all eukaryotic cells *To whom correspondence should be addressed: Department of Bio- cope with ER stress and maintain the homeostasis of the ER physics, Graduate School of Science, Kyoto University, Kitashirakawa- by activating the unfolded protein response (UPR) (Ron and Oiwake, Sakyo-ku, Kyoto 606-8502, Japan. Walter, 2007). Tel: +81–75–753–4067, Fax: +81–75–753–3718 UPR signaling is transduced across the ER membrane by E-mail: [email protected] Abbreviations: A1AT, α1-antitrypsin; CFP, cyan-emitting green fluores- a transmembrane protein present in the ER (Mori, 2000). cent protein; DIG, digoxigenin; eIF2α, α subunit of eukaryotic translation The budding yeast Saccharomyces cerevisiae expresses initiation factor 2; ER, endoplasmic reticulum; GFP, green fluorescent pro- Ire1p, a transmembrane protein kinase/endoribonuclease in tein; KO, knockout; MEFs, mouse embryonic fibroblasts; tsVSVG, tem- perature-sensitive vesicular stomatitis virus G protein; UPR, unfolded the ER, which upon ER stress initiates unconventional protein response; WT, wild-type. splicing of HAC1 mRNA. This in turn results in production 75 Y. Adachi et al. of the UPR-specific transcription factor Hac1p, leading to mice as well as MEFs deficient in ATF6α, and reached the transcriptional induction of hundreds of genes encoding similar but not identical conclusions (Wu et al., 2007) (see proteins working at various stages of secretion, including the Discussion for details). both ER chaperones and ERAD components. Induced pro- Our previous analysis focused on selected canonical tar- teins help yeast cells to deal with unfolded proteins accumu- get genes of the UPR. Here, to unambiguously clarify the lated in the ER (see the Discussion for details). Importantly, role of ATF6α, we performed a genome-wide search for the number of such UPR transducers has increased with ATF6α-target genes. Based on the results, we propose that evolution, allowing higher organisms to cope with ER stress ATF6 is a transcription factor which is specialized in the in a more sophisticated way (Bernales et al., 2006). regulation of ER quality control proteins. Mammalian ER expresses three transmembrane UPR transducers, which carry characteristic effector domains in their cytoplasmic regions (Schroder and Kaufman, 2005). Experimental Procedures These are IRE1 (Ire1p homologue), PERK (transmembrane protein kinase) and ATF6 (transmembrane transcription Preparation, culture and transfection of ATF6α+/+ and factor). Thus, in contrast to yeast cells, which cope with ER ATF6α–/– MEFs stress only by inducing transcription, mammalian cells are capable of decreasing the burden on the ER by attenuating Male heterozygotes of ATF6α (ATF6α+/–) (Yamamoto et translation generally via the activation of PERK, which al., 2007) were backcrossed to female wild-type mice phosphorylates the α subunit of eukaryotic translation initi- (C57BL/6J) eight times to obtain ATF6α N8-heterozygotes. ation factor 2 (eIF2α) (Ron, 2002). In addition, mammalian Crosses between male and female ATF6α N8-heterozygotes cells are capable of inducing the transcription of a variety of were dissected on embryonic day 13.5 and MEFs were iso- sets of genes by activating three transcription factors down- lated by trypsinization of embryos. Primary N8-MEFs were stream of the three UPR transducers. Activated PERK- cultured in Dulbecco’s modified Eagle’s medium (glucose mediated translational attenuation paradoxically induces the at 4.5 g/liter) supplemented with 10% fetal bovine serum, translation of transcription factor ATF4 (Harding et al., 2 mM glutamine, and antibiotics (100 U/ml penicillin and 2000a). Activated IRE1 initiates unconventional splicing of 100 µg/ml streptomycin) at 37°C in a humidified 5% CO2/ XBP1 mRNA to produce the highly active transcription 95% air atmosphere. Transfection was performed using factor pXBP1(S), a functional homologue of yeast Hac1p FuGENE6 (Roche) according to the manufacturer’s instruc- (Calfon et al., 2002; Yoshida et al., 2001a). ATF6 is con- tions. pECFP-N1-tsVSVG and pECFP-N1-A1AT to express verted to an active transcription factor by ER stress-induced tsVSVG-CFP and A1AT-CFP fusion proteins, respectively, regulated intramembrane proteolysis (Mori, 2003). A full were as described previously (Nadanaka et al., 2004). understanding of the molecular mechanisms and biological PERK+/+ and PERK–/– MEFs (Harding et al., 2000b) were significance of the mammalian UPR requires that both the the generous gift of Dr. David Ron (New York University). differential as well as overlapping roles of these three tran- XBP1+/+ and XBP1–/– MEFs (Lee et al., 2003) were the scriptional induction pathways be determined. generous gift of Dr. Laurie Glimcher (Harvard Medical ATF6, consisting of the closely related ATF6α and School). ATF6β in mammals, is constitutively synthesized as a type II transmembrane protein in the ER, designated pATF6α/ Microarray analysis β(P) (Haze et al., 2001; Haze et al., 1999). Upon ER stress, pATF6α/β(P) relocates from the ER to the Golgi apparatus Total RNA extracted from ATF6α+/+ and ATF6α–/– N8- to be cleaved by the sequential action of site-1 and site-2 MEFs by the acid guanidinium/phenol/chloroform method proteases (Nadanaka et al., 2004; Shen et al., 2002a; Ye using ISOGEN (Nippon Gene) was further purified using et al., 2000). The resulting cytoplasmic fragment liberated RNeasy Mini (Qiagen), and checked for quality with an from the membrane, designated pATF6α/β(N), enters the RNA 6000 Nano Assay using an Agilent 2100 Bioanalyser nucleus to activate transcription of its target genes (Yoshida (Agilent Technologies). Five hundred nanogram aliquots of et al., 2000; Yoshida et al., 2001b). We have recently gener- total RNA prepared from N8-MEFs untreated or treated ated ATF6α- and ATF6β-knockout mice, which developed with 2 µg/ml tunicamycin for 8 h were converted to cDNA normally, and found that their double knockout caused by reverse transcription. cDNA obtained from untreated and embryonic lethality (Yamamoto et al., 2007). Analysis of tunicamycin-treated N8-MEFs was then labeled by tran- mouse embryonic fibroblasts (MEFs) deficient in either scription with cyanine