CELL STRUCTURE AND FUNCTION 33: 75–89 (2008) © 2008 by Japan Society for Cell Biology

ATF6 Is a Specializing in the Regulation of Quality Control in the

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 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 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 ; 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 ) 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 3-CTP and cyanine 5-CTP, respec- ATF6α or ATF6β showed that ATF6α but not ATF6β tively, using an Agilent Low Input Linear Amplification kit. is required for transcriptional induction of not only ER After purification through RNeasy Mini, 825 ng each of chaperones but also ERAD components, and that the labeled cRNA probes was mixed and hybridized with a ATF6α–/– MEFs are sensitive to ER stress. Wu et al. inde- 4×44K Agilent oligo microarray (Whole Mouse Genome), pendently generated and characterized ATF6α-knockout on which 44,000 mouse genes were spotted, using an

76 ATF6 as a Specialized Transcription Factor for ER Quality Control

Agilent Hybridization Kit. Cyanine 3 and tunicamycin, an inhibitor of protein N-glycosylation known cyanine 5 fluorescence intensities of a spot were obtained to evoke ER stress (Kaufman, 1999), we conducted four after subtraction of respective background intensity of the independent microarray analyses, and obtained fold- spot using a GenePix 4000B (Axon). Fold induction caused induction values for 14,729 of 44,000 spotted mouse genes by tunicamycin treatment was defined as the ratio of at least three and mostly four times (see Experimental Proce- cyanine 5 intensity to cyanine 3 intensity. Because our anal- dures). The number of genes induced more than 2-fold by ysis was based on fold induction values, genes showing tunicamycin treatment was 680 and 556 in ATF6α+/+ extremely low fluorescence intensity were eliminated to and ATF6α–/– MEFs, respectively. ATF6α targets were ensure accuracy. To this end, background intensity obtained defined as genes whose fold induction value in ATF6α+/+ for 44,000 spots was summed and the average background MEFs was more than 2 and in ATF6α–/– MEFs was less intensity was determined for both cyanine 5 and cyanine 3: than half that in ATF6α+/+ MEFs. This process produced a if cyanine 5 or cyanine 3 intensity for a spot was less than total of 30 genes as ATF6α-target genes (Table I), which half the respective average background intensity, the fold were categorized into several functional groups as detailed induction value was not determined. We carried out four below. independent experiments and obtained fold induction values for 14,729 of 44,000 genes at least three and mostly four Requirement of ATF6α for transcriptional induction of times. most ER chaperones

Northern blot hybridization Microarray analysis showed that mRNA encoding the major ER chaperone BiP (Sitia and Braakman, 2003) was induced Total RNA was extracted from cultured N8-MEFs using 11-fold in ATF6α+/+ MEFs in response to tunicamycin ISOGEN. Northern blot hybridization was performed treatment (Fig. 1A). This induction was mitigated to less than according to standard procedures (Sambrook et al., 1989). 5-fold in ATF6α–/– MEFs. Similarly, the transcriptional Digoxigenin (DIG)-labeled cDNA probes were prepared induction of ER-localized molecular chaperones ORP150/ using PCR according to the manufacturer’s instructions GRP170, GRP94 and calreticulin (Sitia and Braakman, (Roche) and hybridized with RNA electrophoresed and 2003) observed in ATF6α+/+ MEFs was greatly mitigated blotted on a membrane. Subsequent reaction with anti- in ATF6α–/– MEFs (Fig. 1A). These results are consistent digoxigenin antibody (Roche) and treatment with the with our previous northern blot hybridization analysis of chemiluminescent detection reagent CDP-star (GE Health- BiP as well as immunoblotting analysis of BiP and GRP94 care Biosciences) were performed according to the manu- (Yamamoto et al., 2007), demonstrating that ATF6α is gen- facturer’s specifications. Chemiluminescence was visualized erally required for transcriptional induction of ER-localized using an LAS-3000mini LuminoImage analyzer (Fuji Film). molecular chaperones, with the possible exception of calnexin (Sitia and Braakman, 2003) and STCH (Otterson Immunological techniques et al., 1994). Transcriptional induction of GRP75, a mitochon- drial molecular chaperone (Szabadkai et al., 2006), did Immunoblotting analysis was carried out according to the not depend on ATF6α, as expected. standard procedure (Sambrook et al., 1989) as described ERp72, P5 and GRP58 are protein disulfide isomerase- previously (Okada et al., 2002) using Western Blotting like folding enzymes containing thioredoxin motifs (Sitia Luminol Reagent (Santa Cruz Biotechnology). Chemi- and Braakman, 2003). As shown in Fig. 1B, transcriptional luminescence was detected using an LAS-3000mini Lumino- induction of these three enzymes observed in ATF6α+/+ Image analyzer (Fuji Film). ATF6α was detected with MEFs was lost or greatly mitigated in ATF6α–/– MEFs. rabbit anti-ATF6α polyclonal antibody (Haze et al., 1999). After oxidization of substrates, protein disulfide isomerase Anti-GFP monoclonal antibody (mixture of clone 7.1 and is reoxidized by the action of Ero1, which consists of 13.1) was purchased from Roche. Mouse anti-KDEL anti- closely related ERO1α and ERO1β in mammals (Sitia and body was purchased from Stressgen. Immunoprecipitation Braakman, 2003). Microarray analysis showed that weak was carried out essentially as described previously (Nadanaka transcriptional induction of ERO1α occurred similarly in et al., 2004). ATF6α+/+ and ATF6α–/– MEFs, whereas the marked tran- scriptional induction of ERO1β observed in ATF6α+/+ MEFs was lost in ATF6α–/– MEFs (Fig. 1B). These obser- Results vations were well confirmed by northern blot hybridization analysis of MEFs treated with tunicamycin or thapsigargin, 2+ Identification of ATF6α-target genes an inhibitor of ER-Ca -ATPase known to evoke ER stress (Kaufman, 1999), as shown in Fig. 1C. Using total RNA isolated from ATF6α+/+ and ATF6α–/– p58IPK (Rutkowski et al., 2007), ERdj3 (Shen and MEFs which had been untreated or treated for 8 h with Hendershot, 2005) and ERdj4 (Shen et al., 2002b) are DnaJ

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Table I. LIST OF ATF6α-TARGET GENES

WT KO Gene title (Function) Location P value Fold induction SD Fold induction SD ER chaperones ERO1β 11.27 0.63 1.28 0.76 <0.01 BiP 11.11 1.99 4.87 0.99 <0.01 p58IPK 7.13 0.17 2.03 0.42 <0.01 ORP150/GRP170 6.27 0.56 2.06 0.36 <0.01 GRP94 4.65 0.38 1.89 0.33 <0.01 ERp72 2.97 0.33 1.05 0.23 <0.01 ERdj3 2.70 0.27 1.07 0.14 <0.01 (P5) 2.45 0.29 1.50 0.14 <0.01 (GRP58) 1.95 0.07 1.05 0.07 <0.01 (CRT) 1.78 0.20 0.95 0.13 <0.01 ERAD components Derlin-3 20.14 2.03 1.44 0.83 <0.01 Herp 11.61 2.06 2.92 0.96 <0.01 SEL1L 5.42 1.09 1.30 0.31 <0.01 HRD1 3.89 0.35 1.28 0.22 <0.01 EDEM1 2.66 0.97 1.28 0.61 <0.01 ER proteins SDF2L1 Protein O-glycosylation SP+ 10.25 1.86 2.40 0.76 <0.01 CRELD2 SP+ 8.00 2.09 1.25 0.30 <0.05 ARMET SP+ 5.79 0.72 2.58 0.47 <0.01 PIG-A GPI anchor biosynthesis TM1 5.55 0.75 1.41 0.17 <0.01 SERCA2 Ca2+ ion transporter TM8 3.59 0.30 1.68 0.28 <0.01 ORMDL2 Protection from ER stress TM3 3.46 0.81 0.97 0.26 <0.01 Protein glycosylation Galnt3 Protein O-glycosylation Golgi, TM1 2.17 0.28 1.04 0.09 <0.01 Transcription factor homeo box A1 (Hoxa1) Nucleus 5.01 1.57 1.82 0.55 * Lipid metabolism Mlstd2/FAR1 Fatty acyl CoA reductase 1 Peroxisome, TM1 2.12 0.21 0.85 0.18 <0.01 DNA binding nucleobindin 2 Extracellular, SP+ 2.93 0.72 1.43 0.28 <0.05 Ca2+ ion binding calmodulin 1 Cytoplasm 2.16 0.88 1.01 0.18 * Cell adhesion CEACAM20 ? (SP+, TM1) 2.58 0.26 0.99 0.16 * Unknown function Ccdc134, coiled-coil domain containing 134 ? (SP+) 3.33 0.67 1.43 0.28 <0.05 RIKEN cDNA 2810026P18 gene ? 3.00 0.53 1.39 0.76 * RIKEN cDNA 2900034E22 gene ? 2.96 0.58 1.36 0.57 <0.05 Hrg, histidine-rich glycoprotein Plasma membrane, SP+ 2.45 1.37 0.57 0.16 * lin-52 (C. elegans) homolog ? (SP–) 2.43 0.61 1.10 0.39 <0.05 Tbccd1, TBCC domain containing 1 ? (SP–) 2.26 0.46 1.12 0.13 <0.05 Three genes in parenthesis are not ATF6α-target genes in our criteria but are shown for reference. SP, signal peptide TM, transmembrane domain *statistically insignificant

78 ATF6 as a Specialized Transcription Factor for ER Quality Control

Fig. 1. Effect of the absence of ATF6α on transcriptional induction of molecular chaperones. (A) ATF6α+/+ (WT, open bars) and ATF6α–/– (KO, closed bars) MEFs were untreated or treated with 2 µg/ml tunicamycin for 8 h. Fold induction of various molecular chaperones indicated on the abscissa was determined by microarray analysis and is shown as the means ± S.D. of four independent experiments. P<0.01 for the difference in induction of BiP, ORP150/GRP170, GRP94 and calreticulin (CRT) between ATF6α+/+ and ATF6α–/– MEFs. There was no statistically significant difference in the induction of calnexin (CNX), STCH or GRP75. (B) Fold induction of various folding enzymes indicated on the abscissa was determined and is shown as in (A). P<0.01 for the difference in induction of ERp72, P5, GRP58 and ERO1β. There was no statistically significant difference in the induction of ERO1α. (C) ATF6α+/+ and ATF6α–/– MEFs were treated with 2 µg/ml tunicamycin (Tm, left panel) or 300 nM thapsigargin (Tg, right panel) for the indicated periods. Total RNA was isolated and analyzed by northern blot hybridization using a DIG-labeled cDNA probe specific to mouse BiP, ERO1α, ERO1β or GAPDH.

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Fig. 2. Effect of the absence of ATF6α on transcriptional induction of ER cochaperones. (A) Fold induction of three ER cochaperones was determined and is shown as in Fig. 1A. P<0.01 for the difference in induction of p58IPK, ERdj3 and ERdj4. (B) ATF6α+/+ and ATF6α–/– MEFs were treated with 2 µg/ml tunicamycin (Tm). Total RNA was isolated and analyzed by northern blot hybridization using a DIG-labeled cDNA probe specific to mouse BiP, ERdj3, ERdj4 or GAPDH. domain-containing cochaperones inducible by ER stress. SEL1L, HRD1, EDEM1, known components of ERAD Microarray analysis showed that the induction of p58IPK (Kawaguchi and Ng, 2007), largely depended on ATF6α, and ERdj3 mRNA observed in ATF6α+/+ MEFs was lost or consistent with our previous northern blot hybridization greatly mitigated in ATF6α–/– MEFs (Fig. 2A), consistent analysis (Yamamoto et al., 2007). p97/VCP, a cytosolic with our previous northern blot hybridization analysis AAA-ATPase which plays a key role in extracting ERAD (Yamamoto et al., 2007). In contrast, microarray analysis substrates (Ye et al., 2001), was not inducible during ER showed that induction of ERdj4 mRNA in ATF6α–/– MEFs stress. In contrast, transcriptional induction of RAMP4, an was nearly half that in ATF6α+/+ MEFs (Fig. 2A), though ER membrane protein of unknown function (Hori et al., our previous northern blot hybridization analysis showed 2006), occurred similarly in ATF6α+/+ and ATF6α–/– that induction of ERdj4 mRNA occurred similarly in MEFs (Fig. 3A), consistent with our previous northern blot ATF6α+/+ and ATF6α–/– MEFs (Yamamoto et al., 2007). hybridization analysis (Yamamoto et al., 2007). Similarly, The only difference here is that our previous northern blot transcriptional induction of Derlin-1 and Derlin-2, proteins hybridization analysis was conducted with MEFs isolated that span the ER membrane four times and are required for from N1 mice, whereas our microarray analysis was con- ERAD (Lilley and Ploegh, 2004; Oda et al., 2006; Ye et al., ducted with MEFs isolated from N8 mice. We therefore 2004), was not significantly affected by the absence of performed northern blot hybridization analysis of N8-MEFs ATF6α (Fig. 3A). This observation was confirmed by and found that induction patterns of ERdj3 and ERdj4 were northern blot hybridization as shown in Fig. 3B. Further, identical to those previously reported in N1-MEFs, as VIMP and EDEM3, known components of ERAD shown in Fig. 2B, indicating that the inconsistent results of (Kawaguchi and Ng, 2007), also did not depend on ATF6α microarray analysis of ERdj4 were within the acceptable for induction (Fig. 3A). Interestingly, marked induction of range of experimental error. Derlin-3, a protein more closely related to Derlin-2 than to Proteins analyzed in Fig. 1 and Fig. 2 represent all ER- Derlin-1 (Oda et al., 2006), absolutely required ATF6α localized molecular chaperones, cochaperones and folding (Fig. 3A), and this observation was firmly confirmed by enzymes induced by tunicamycin treatment in our microarray northern blot hybridization analysis (Fig. 3B). As the pro- analysis. From these, we thus concluded that ATF6α is re- teins shown in Fig. 3A are all known ERAD components quired for transcriptional induction of most ER chaperones. which we found inducible during tunicamycin treatment in our microarray analysis, we concluded that ATF6α is Requirement of ATF6α for transcriptional induction of required for transcriptional induction of some but not all some ERAD components ERAD components. As shown in Fig. 3A, transcriptional induction of Herp,

80 ATF6 as a Specialized Transcription Factor for ER Quality Control

Fig. 3. Effect of the absence of ATF6α on transcriptional induction of ERAD components. (A) Fold induction of various ERAD components indicated on the abscissa was determined and is shown as in Fig. 1A. P<0.01 for the difference in induction of Herp, SEL1L, HRD1, EDEM1, and Derlin-3. P<0.05 for the difference in induction of Derlin-2 and Derlin-1. There was no statistically significant difference in the induction of p97/VCP, RAMP4, VIMP or EDEM3. (B) ATF6α+/+ and ATF6α–/– MEFs were treated and analyzed as in Fig. 1C using a DIG-labeled cDNA probe specific to mouse BiP, Derlin-1, Derlin-2, Derlin-3 or GAPDH.

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Cross talk among UPR mediators As shown in Fig. 4A, the absence of ATF6α had almost no effect on expression levels of two other UPR transducers, IRE1α and PERK. Transcriptional induction of XBP1 and ATF4, downstream transcription factors of the IRE1 and PERK pathways, respectively, occurred similarly in ATF6α+/+ and ATF6α–/– MEFs, consistent with our previ- ous northern blot hybridization and immunoblotting analy- ses (Yamamoto et al., 2007). The transcription factor CHOP is known to be regulated by both ATF6 and PERK path- ways (Ma et al., 2002; Okada et al., 2002). Accordingly, the transcriptional induction of CHOP observed in ATF6α+/+ MEFs was mitigated considerably in ATF6α–/– MEFs (Fig. 4A). Transcriptional induction of GADD34 (Novoa et al., 2001) and asparagine synthetase (Barbosa-Tessmann et al., 2000), known targets of the PERK pathway, was not affected by the absence of ATF6α, as expected. It was previously shown that transcriptional induction of ER chaperones was significantly mitigated in MEFs defi- cient in PERK (Harding et al., 2000a; Wu et al., 2007) or in MEFs in which the phosphorylation site of eIF2α had been replaced by alanine (Scheuner et al., 2001), but that the downstream transcription factor ATF4 does not bind to the cis-acting ER stress-response element responsible for induc- tion of ER chaperones (Ma et al., 2002). We therefore examined the effects of the absence of PERK on activation of ATF6α. As shown in Fig. 4B, pATF6α(P) was cleaved to produce pATF6α(N) 2 h after treatment with tunicamycin in both PERK+/+ (lane 2) and PERK–/– (lane 8) MEFs, but this activation was not sustained in PERK–/– MEFs (lanes 9–12) in contrast to the case of PERK+/+ MEFs (lanes 3–6). Accordingly, BiP was less induced in PERK–/– MEFs than in PERK+/+ MEFs. These findings indicated that the absence of PERK exerted its inhibitory activity on the induction of ER chaperones via the blocking of ATF6α acti- vation, the mechanism of which is currently under investi- gation.

Dispensability of ATF6α in transcriptional induction of α proteins involved in translocation into and transport Fig. 4. Effect of the absence of ATF6 on the levels of two other UPR transducers and transcriptional induction of their downstream proteins, as from the ER well as effect of the absence of PERK on activation of ATF6α. (A) Fold induction of UPR transducers and their downstream proteins indicated on A number of proteins work together for the translocation of the abscissa was determined and is shown as in Fig. 1A. P<0.05 for the newly synthesized secretory and transmembrane proteins difference in induction of CHOP. There was no statistically significant into the ER as well as their transport from the ER to the difference in the induction of XBP1, ATF4, PERK, IRE1α, GADD34 or Golgi apparatus, some of which are known to be upregu- asparagine synthetase (ASN-S). (B) PERK+/+ and PERK–/– MEFs were lated during the UPR. We therefore examined whether these treated with 10 µg/ml tunicamycin (Tm) for the indicated periods. Cell translocation and transport proteins are regulated by lysates were prepared and analyzed by immunoblotting using anti-ATF6α α ATF6α. As shown in Fig. 5A, microarray analysis revealed or anti-KDEL antibody. The migration positions of pATF6 (P), pATF6α(P*), pATF6α(N) and BiP are marked. pATF6α(P*) denotes the that SRP54 (Bernstein et al., 1989) showed the highest nonglycosylated form of pATF6α(P). The positions of full-range rainbow inducibility among the various proteins involved in translo- molecular weight markers (GE Healthcare Biosciences) are indicated on cation into the ER and that its induction was not affected by the left. the absence of ATF6α. This observation was well con- firmed by northern blot hybridization analysis (Fig. 5C):

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Fig. 5. Effect of the absence of ATF6α on transcriptional induction of proteins involved in translocation into the ER and transport from the ER. (A) Fold induction of various proteins involved in translocation into the ER indicated on the abscissa was determined and is shown as in Fig. 1A. There was no statistically significant difference in the induction of any of the 9 genes. (B) Fold induction of various proteins involved in transport from the ER to the Golgi apparatus indicated on the abscissa was determined and is shown as in Fig. 1A. P<0.01 and P<0.05 for the difference in induction of VDP and SAR1a, respectively. There was no statistically significant difference in the induction of the other 6 genes. (C) ATF6α+/+ and ATF6α–/– MEFs were treated and analyzed as in Fig. 1C using a DIG-labeled cDNA probe specific to mouse BiP, SRP54, SEC23b, VDP, BET1 or GAPDH.

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Fig. 6. Effect of the absence of XBP1 on transcriptional induction of proteins involved in translocation into the ER and transport from the ER. XBP1+/+ and XBP1–/– MEFs were treated and analyzed as in Fig. 1C using a DIG-labeled cDNA probe specific to mouse BiP, SRP54, SEC23b, VDP, BET1 or GAPDH. induction of BiP mRNA as a control was greatly mitigated temperature of 32°C, in contrast, it is rapidly folded and in ATF6α–/– MEFs as compared with ATF6α+/+ MEFs. then transported to the plasma membrane through the Golgi Further, transcriptional induction of several other proteins apparatus (Nehls et al., 2000). As shown in Fig. 7A, after involved in translocation was also not dependant on temperature downshift to 32°C, tsVSVG fusing to cyan- ATF6α, although the extent of their induction was low. emitting green fluorescent protein (tsVSVG-CFP) showed Similarly, three proteins involved in the ER-Golgi trans- doublet protein bands (upper panel), which were resolved port, namely SEC23b (Paccaud et al., 1996), VDP (Allan more clearly when samples were treated with endo- et al., 2000) and BET1 (Zhang et al., 1997), were also glycosidase H (lower panel). As the endoglycosidase H- induced in ATF6α+/+ and ATF6α–/– MEFs, as determined resistant form (upper migrating band) represents the protein by microarray analysis (Fig. 5B) and confirmed by northern modified at the Golgi apparatus, tsVSVG-CFP moved to blot hybridization analysis (Fig. 5C). Importantly, transcrip- the Golgi apparatus with similar kinetics in ATF6α+/+ tional induction of SEC23b, VDP and BET1 as well as and ATF6α–/– MEFs. SRP54 required XBP1 as their mRNA was induced in We also examined whether secretion of α1-antitrypsin XBP1+/+ MEFs but not in XBP1–/– MEFs (Fig. 6); BiP (A1AT), a serum glycoprotein, was affected by the absence mRNA as a control was induced similarly in XBP1+/+ and of ATF6α. We expressed A1AT-CFP fusion protein by XBP1–/– MEFs, as reported previously (Lee et al., 2003; transfection and found that A1AT-CFP was secreted simi- Lee et al., 2002). These results indicated that ATF6 is not larly in ATF6α+/+ and ATF6α–/– MEFs even in the pres- required for transcriptional induction of either translocation ence of 1 mM dithiothreitol, a reducing reagent known to proteins or transport proteins. cause ER stress (Kaufman, 1999), as shown in Fig. 7B: as We finally examined whether the absence of ATF6α A1AT contains only one cysteine residue, dithiothreitol affects the transport of cargo proteins from the ER to the should have no effect on the folding of A1AT (Nadanaka et Golgi apparatus using a temperature-sensitive mutant of al., 2004). In ATF6α+/+ or ATF6α–/– MEFs treated with vesicular stomatitis virus G protein (tsVSVG) as a model tunicamycin, in contrast, A1AT was not secreted and its protein. At the non-permissive temperature of 39.5°C, deglycosylated and thus misfolded form was accumulated tsVSVG is misfolded due to a point mutation in its luminal intracellularly (Fig. 7B). These results indicated that cargo domain and retained in the ER. After shift to the permissive proteins are transported from the ER normally in ATF6α–/–

84 ATF6 as a Specialized Transcription Factor for ER Quality Control

Fig. 7. Effect of the absence of ATF6α on transport of cargo proteins from the ER. (A) ATF6α+/+ and ATF6α–/– MEFs were transfected with pECFP- N1-tsVSVG to express tsVSVG-CFP fusion protein. Four hours later, transfected MEFs were incubated at the non-permissive temperature of 39.5°C for 18 h and then at the permissive temperature 32°C for the indicated periods in the absence (–) or presence (+) of 1 mM dithiothreitol (DTT). Cell lysates were prepared, treated with (+) or without (–) endoglycosidase H (Endo H), and analyzed by immunoblotting using anti-GFP monoclonal antibody. Migration positions of tsVSVG-CFP, a mixture of Endo H-resistant and -sensitive forms (upper panel) as well as the endo-H resistant form, tsVSVG-CFP (mature), and the deglycosylated form, tsVSVG-CFP (-CHO), (lower panel) are shown. (B) ATF6α+/+ and ATF6α–/– MEFs were transfected with pECFP-N1-A1AT to express A1AT-CFP fusion protein. Seventeen hours later, transfected MEFs were fed with fresh medium and then incubated at 37°C for the indicated periods in the presence of 1 mM dithiothreitol (DTT) or 2 µg/ml tunicamycin (Tm). Cell lysates were prepared and analyzed by immunoblotting using anti-GFP monoclonal antibody, whereas medium was collected and subjected to immunoprecipitation using anti-GFP monoclonal antibody. Migration positions of A1AT-CFP as well as monoclonal antibody (loading control) are shown. A1AT-CFP* denotes the nonglycosylated form of A1AT-CFP.

MEFs if they are folded correctly. We concluded that trans- chaperone BiP) and GRP94 (found to be a molecular port from the ER to the Golgi apparatus was not affected by chaperone of the Hsp90 family) (Kozutsumi et al., 1988). the absence of ATF6α. This finding in turn implied that the UPR is a homeostatic response which maintains the protein folding environment in the ER by suppressing the proteotoxicity of accumulated Discussion unfolded proteins. The use of the budding yeast Saccharomyces cerevisiae as a model in the 1990s led to major progress in A prototype of the UPR was first discovered in the 1970s, understanding the molecular mechanisms of the UPR, and when analysis of virus-transformed mammalian cells identi- to molecular cloning of the UPR transducer Ire1p present in fied GRP78 and GRP94 as proteins inducible by glucose the ER (Cox et al., 1993; Mori et al., 1993), as well as the starvation (Shiu et al., 1977). Substantial analysis of UPR-specific transcription factor Hac1p (Cox and Walter, the UPR began in the late 1980s, when the accumulation 1996; Mori et al., 1996); followed by discovery of Ire1p- of unfolded proteins in the ER was found to trigger the mediated splicing of HAC1 mRNA, which connects the event induction of GRP78 (found to be identical to the major ER in the ER to that in the nucleus (Cox and Walter, 1996;

85 Y. Adachi et al.

Kawahara et al., 1997). A number of ER chaperones were In the present study, we conducted a genome-wide search identified as targets of the Ire1p-Hac1p pathway in the yeast for ATF6α-target genes by microarray analysis of MEFs UPR (Mori et al., 1998). Importantly, microarray analysis, a deficient in ATF6α. Among the 14,729 mouse genes we new technology invented in the late 1990s, increased the could successfully analyze, 680 and 556 genes were number of UPR target genes drastically, to 381 out of a total induced more than 2-fold in response to tunicamycin treat- 6,000 yeast genes, including not only ER chaperones but ment of ATF6α+/+ and ATF6α–/– MEFs, respectively. also numerous proteins working at various stages of secre- When the following criteria were applied, however, only 30 tion, specifically proteins involved in translocation, protein genes were identified as ATF6α-target genes: the ATF6α- folding, protein degradation, glycosylation in the ER, target gene had to be induced more than 2-fold in ATF6α+/+ lipid/inositol metabolism, ER-Golgi transport, Golgi-ER MEFs and its fold induction value in ATF6α–/– MEFs had retrieval, glycosylation in the Golgi apparatus, vacuolar tar- to be less than half that in ATF6α+/+ MEFs. Among these geting, distal secretion and cell wall biogenesis. Based on 30 genes, notably, 7 were ER chaperones, 5 were ERAD these findings, it was proposed that activation of the yeast components and 6 were ER proteins; six ER proteins are UPR induces specific remodeling of the secretory pathway SDF2L1 for protein O-glycosylation (Fukuda et al., 2001); to minimize the amount, concentration, or both of unfolded CRELD2 with unknown function (Ortiz et al., 2005); proteins in the ER (Travers et al., 2000). ARMET with unknown function (Mizobuchi et al., 2007); In metazoan cells, the UPR transducer Ire1p is well con- PIG-A for glycosylphosphatidylinositol anchor biosynthesis served, whereas the target transcription factor of Ire1p- (Watanabe et al., 1996); SERCA2, Ca2+-ATPase (Thuerauf mediated unconventional splicing is switched from Hac1p et al., 2001); and ORMDL2 for protection from ER stress to XBP1 (Calfon et al., 2002; Shen et al., 2001; Yoshida (Hjelmqvist et al., 2002) (Table I). The remaining 12 targets et al., 2001a). The IRE1-XBP1 pathway is responsible for in- were miscellaneous and could not be further categorized. duction of most canonical UPR target genes in C. elegance ATF6α therefore appears to have limited function as com- (Shen et al., 2005) and D. melanogaster (Hollien and pared with XBP1, and may regulate ER quality control pro- Weissman, 2006). Interestingly, however, the IRE1-XBP1 teins primarily and specifically. pathway is dispensable to the induction of major ER chaper- It is particularly noteworthy that proteins involved in ones such as GRP78/BiP and GRP94 in mammalian cells, translocation into the ER (SRP54, SSR3 and SPCS3) as but is required for induction of a subset of ER chaperones well as ER-Golgi transport (SEC23b, VDP and BET1) are and most ERAD components (Lee et al., 2003; Lee et al., not ATF6α targets, but rather XBP1-targets (Figs. 5 and 6). 2002; Oda et al., 2006; Yoshida et al., 2003), thus revealing We did not observe any defects in the transport of cargo diversity in the transcriptional induction system in mam- proteins (tsVSVG and A1AT) out of the ER to the plasma mals. Extensive genome-wide searches using microarray or membrane or extracellular space via the Golgi apparatus in chromatin immunoprecipitation analyses by several labo- ATF6α–/– MEFs as compared with ATF6α+/+ MEFs (Fig. ratories consistently showed that mammalian XBP1 is 7). Wu et al. independently generated ATF6α-knockout involved in the expression of not only genes working for mice and characterized ATF6α-deficient MEFs (Wu et al., protein folding and degradation in the ER but also many 2007). Their conclusions were similar to ours as far as regu- genes working for the secretory pathway and others, simi- lation of ER chaperones and ERAD components are con- larly to the case of S. cerevisiae, indicating that XBP1 is a cerned. Nonetheless, they found that transport of transferin multifunctional transcription factor (Acosta-Alvear et al., from the ER to the Golgi apparatus or secretion of 2007; Lee et al., 2003; Shaffer et al., 2004; Sriburi et al., alkaline phosphatase into the extracellular space was ineffi- 2007). cient in ATF6α–/– MEFs treated with thapsigargin as com- The UPR transducer ATF6 is present in metazoan pared with untreated ATF6α–/– MEFs or ATF6α+/+ MEFs genomes but not in the yeast genome. Worm and fly treated with thapsigargin, leading them to propose that genomes contain a single ATF6 gene, whereas mammalian ATF6α is required to optimize protein folding, secretion genomes contain two closely related genes, ATF6α and and degradation during ER stress. In their microarray analy- ATF6β. Although worm and fly ATF6 has little role in the sis, however, only Rab38 was identified as an ATF6α-target UPR (Hollien and Weissman, 2006; Shen et al., 2005), we among numerous proteins involved in vesicular transport if recently showed that mammalian ATF6α but not ATF6β our criteria for ATF6α-target genes were applied, and Rab38 is required for transcriptional induction of major ER appeared to control post-Golgi trafficking (Wasmeier et al., chaperones as well as of ERAD components. Detailed 2006). In our analysis, signals of Rab38 were extremely analysis showed that ATF6α is solely responsible for the weak and thus Rab38 was excluded from the 14,729 genes transcriptional induction of major ER chaperones and that we analyzed. Therefore, the molecular basis of their pro- ATF6α heterodimerizes with XBP1 for the induction of posal regarding secretion defects of ATF6α–/– MEFs seems major ERAD components (Yamamoto et al., 2007). Thus, unsupported. Rather, we suspect that the inefficient trans- ATF6 has gained the ability to induce the canonical UPR port of transferin receptor or alkaline phosphatase in target genes in higher eukaryotes. ATF6α–/– MEFs treated with thapsigargin as compared

86 ATF6 as a Specialized Transcription Factor for ER Quality Control with similarly treated ATF6α+/+ MEFs may be due to fold- Acknowledgements. We are grateful to Dr. D. Ron for his provision of ing defects resulting from inefficient induction of ER PERK+/+ and PERK–/– MEFs and Dr. L. Glimcher for XBP1+/+ and chaperones. It is markedly difficult to separate transport XBP1–/– MEFs. We thank Ms. Kaoru Miyagawa for her technical and secretarial assistance. This work was supported in part by grants from the defects from folding defects experimentally, because only Ministry of Education, Culture, Sports, Science and Technology of Japan correctly folded cargo proteins are transported. In our (15GS0310 and 19058009 to K. M.). experiments with tsVSVG (Fig. 7A), transfected cells were incubated at the non-permissive temperature 39.5°C for References 18 h to cause misfolding and retention of tsVSVG in the ER, which should have evoked significant ER stress in both Acosta-Alvear, D., Zhou, Y., Blais, A., Tsikitis, M., Lents, N.H., Arias, C., ATF6α+/+ and ATF6α–/– MEFs. Yet, tsVSVG was trans- Lennon, C.J., Kluger, Y., and Dynlacht, B.D. 2007. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory ported to the Golgi apparatus similarly in ATF6α+/+ and α networks. Mol. Cell, 27: 53–66. ATF6 –/– MEFs after shift to the permissive temperature Allan, B.B., Moyer, B.D., and Balch, W.E. 2000. Rab1 recruitment of 32°C. We therefore concluded that ER-Golgi transport is p115 into a cis-SNARE complex: programming budding COPII vesicles not regulated by ATF6α and that ATF6α is a transcription for fusion. Science, 289: 444–448. factor which is specialized in regulating the expression of Barbosa-Tessmann, I.P., Chen, C., Zhong, C., Siu, F., Schuster, S.M., ER quality control proteins. Nick, H.S., and Kilberg, M.S. 2000. Activation of the human asparagine Based on the present and previously published results, we synthetase gene by the amino acid response and the endoplasmic reticulum stress response pathways occurs by common genomic ele- envision the following scenario to explain the evolution of ments. J. Biol. Chem., 275: 26976–26985. the UPR. First, the IRE1 pathway evolved to counteract the Bernales, S., Papa, F.R., and Walter, P. 2006. Intracellular signaling by the accumulation of unfolded proteins in the ER. Because cells unfolded protein response. Annu. Rev. Cell Dev. Biol., 22: 487–508. such as S. cerevisiae keep synthesizing proteins even under Bernstein, H.D., Poritz, M.A., Strub, K., Hoben, P.J., Brenner, S., and ER stress conditions at this evolutional stage, activated Walter, P. 1989. Model for signal sequence recognition from amino- IRE1 induces transcription of not only ER chaperones and acid sequence of 54K subunit of signal recognition particle. Nature, 340: ERAD components but also numerous proteins working at 482–486. Bertolotti, A., Wang, X., Novoa, I., Jungreis, R., Schlessinger, K., Cho, various stages of secretion to minimize the amount, concen- J.H., West, A.B., and Ron, D. 2001. Increased sensitivity to dextran tration or both of unfolded proteins accumulated in the ER sodium sulfate colitis in IRE1beta-deficient mice. J. Clin. Invest., 107: (Travers et al., 2000). Next, the PERK pathway emerged 585–593. via gene shuffling between IRE1 and GCN2: GCN2 Bukau, B., Weissman, J., and Horwich, A. 2006. Molecular chaperones encodes a protein kinase which phosphorylates eIF2α in and protein quality control. Cell, 125: 443–451. response to amino acid starvation (Silverman and Williams, Calfon, M., Zeng, H., Urano, F., Till, J.H., Hubbard, S.R., Harding, H.P., 1999). Cell such as C. elegance would now be able to atten- Clark, S.G., and Ron, D. 2002. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature, 415: uate translation and decrease the burden on the ER, which 92–96. was perhaps advantageous in handling unfolded proteins in Cox, J.S., Shamu, C.E., and Walter, P. 1993. Transcriptional induction of multicellular organisms, as these encounter not only envi- genes encoding endoplasmic reticulum resident proteins requires a trans- ronmental but also physiological ER stress during develop- membrane protein kinase. Cell, 73: 1197–1206. ment or differentiation. The ATF6 pathway then evolved, Cox, J.S. and Walter, P. 1996. A novel mechanism for regulating activity which is specialized in regulating the transcription of ER of a transcription factor that controls the unfolded protein response. Cell, quality control proteins. Because of the difference between 87: 391–404. Fukuda, S., Sumii, M., Masuda, Y., Takahashi, M., Koike, N., Teishima, J., the IRE1 and ATF6 pathways in their mechanisms of acti- Yasumoto, H., Itamoto, T., Asahara, T., Dohi, K., and Kamiya, K. 2001. vating downstream transcription factors, cells such as those Murine and human SDF2L1 is an endoplasmic reticulum stress-inducible in mammals are able to perform a time-dependent phase gene and encodes a new member of the Pmt/rt protein family. Biochem. shift to cope with ER stress in accordance with the quality, Biophys. Res. Commun., 280: 407–414. quantity, or both of unfolded proteins accumulated in the Harding, H.P., Novoa, I.I., Zhang, Y., Zeng, H., Wek, R., Schapira, M., ER (Yoshida et al., 2003). Finally, various tissue-specific and Ron, D. 2000a. Regulated translation initiation controls stress- UPR transducers, such as IRE1β (Bertolotti et al., 2001) induced gene expression in mammalian cells. Mol. Cell, 6: 1099–1108. Harding, H.P., Zhang, Y., Bertolotti, A., Zeng, H., and Ron, D. 2000b. and ATF6-like membrane-bound transcription factors, i.e. Perk is essential for translational regulation and cell survival during the OASIS (Kondo et al., 2005), CREBH (Zhang et al., 2006), unfolded protein response. Mol. Cell, 5: 897–904. Luman/LZIP (Raggo et al., 2002) and Tisp40 (Nagamori et Haze, K., Okada, T., Yoshida, H., Yanagi, H., Yura, T., Negishi, M., and al., 2005), were developed to strengthen local quality con- Mori, K. 2001. Identification of the G13 (cAMP-response-element- trol capability in the ER. 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Rab38 and Rab32 control post-Golgi trafficking of (Received for publication, December 25, 2007 melanogenic enzymes. J. Cell Biol., 175: 271–281. and accepted, January 31, 2008) Watanabe, R., Kinoshita, T., Masaki, R., Yamamoto, A., Takeda, J., and

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