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∗
1
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan,
2
Laboratory 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
Newly synthesized secretory and transmembrane proteins
are translocated into the endoplasmic reticulum (ER), which
contains a number of molecular chaperones and folding
enzymes (collectively termed ER chaperones hereafter) and
provides an optimal environment for the productive folding
of these proteins. Proteins remaining unfolded or misfolded
even after the assistance of ER chaperones are retrotranslo-
*To whom correspondence should be addressed: Department of Bio-
physics, Graduate School of Science, Kyoto University, Kitashirakawa-
Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
Tel: +81–75–753–4067, Fax: +81–75–753–3718
E-mail: kazu.mori@bio.mbox.media.kyoto-u.ac.jp
Abbreviations: A1AT, α1-antitrypsin; CFP, cyan-emitting green fluores-
cent protein; DIG, digoxigenin; eIF2α, α subunit of eukaryotic translation
initiation factor 2; ER, endoplasmic reticulum; GFP, green fluorescent pro-
tein; KO, knockout; MEFs, mouse embryonic fibroblasts; tsVSVG, tem-
perature-sensitive vesicular stomatitis virus G protein; UPR, unfolded
protein response; WT, wild-type.
cated back to the cytosol, where they are ubiquitinated and
degraded by the proteasome through a process termed ER-
associated degradation (ERAD). These two mechanisms,
productive folding and ERAD, ensure the quality of pro-
teins that pass through the ER and allow only correctly
folded molecules to move along the secretory pathway
(Bukau et al., 2006). However, the ER quality control sys-
tem is compromised under a variety of conditions, collec-
tively termed ER stress, resulting in the accumulation of
unfolded proteins in the ER. Essentially all eukaryotic cells
cope with ER stress and maintain the homeostasis of the ER
by activating the unfolded protein response (UPR) (Ron and
Walter, 2007).
UPR signaling is transduced across the ER membrane by
a transmembrane protein present in the ER (Mori, 2000).
The budding yeast Saccharomyces cerevisiae expresses
Ire1p, a transmembrane protein kinase/endoribonuclease in
the ER, which upon ER stress initiates unconventional
splicing of HAC1 mRNA. This in turn results in production

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of the UPR-specific transcription factor Hac1p, leading to
the transcriptional induction of hundreds of genes encoding
proteins working at various stages of secretion, including
both ER chaperones and ERAD components. Induced pro-
teins help yeast cells to deal with unfolded proteins accumu-
lated in the ER (see the Discussion for details). Importantly,
the number of such UPR transducers has increased with
evolution, allowing higher organisms to cope with ER stress
in a more sophisticated way (Bernales et al., 2006).
Mammalian ER expresses three transmembrane UPR
transducers, which carry characteristic effector domains in
their cytoplasmic regions (Schroder and Kaufman, 2005).
These are IRE1 (Ire1p homologue), PERK (transmembrane
protein kinase) and ATF6 (transmembrane transcription
factor). Thus, in contrast to yeast cells, which cope with ER
stress only by inducing transcription, mammalian cells are
capable of decreasing the burden on the ER by attenuating
translation generally via the activation of PERK, which
phosphorylates the α subunit of eukaryotic translation initi-
ation factor 2 (eIF2α) (Ron, 2002). In addition, mammalian
cells are capable of inducing the transcription of a variety of
sets of genes by activating three transcription factors down-
stream of the three UPR transducers. Activated PERK-
mediated translational attenuation paradoxically induces the
translation of transcription factor ATF4 (Harding et al.,
2000a). Activated IRE1 initiates unconventional splicing of
XBP1 mRNA to produce the highly active transcription
factor pXBP1(S), a functional homologue of yeast Hac1p
(Calfon et al., 2002; Yoshida et al., 2001a). ATF6 is con-
verted to an active transcription factor by ER stress-induced
regulated intramembrane proteolysis (Mori, 2003). A full
understanding of the molecular mechanisms and biological
significance of the mammalian UPR requires that both the
differential as well as overlapping roles of these three tran-
scriptional induction pathways be determined.
ATF6, consisting of the closely related ATF6α and
ATF6β in mammals, is constitutively synthesized as a type
II transmembrane protein in the ER, designated pATF6α/
β(P) (Haze et al., 2001; Haze et al., 1999). Upon ER stress,
pATF6α/β(P) relocates from the ER to the Golgi apparatus
to be cleaved by the sequential action of site-1 and site-2
proteases (Nadanaka et al., 2004; Shen et al., 2002a; Ye
et al., 2000). The resulting cytoplasmic fragment liberated
from the membrane, designated pATF6α/β(N), enters the
nucleus to activate transcription of its target genes (Yoshida
et al., 2000; Yoshida et al., 2001b). We have recently gener-
ated ATF6α- and ATF6β-knockout mice, which developed
normally, and found that their double knockout caused
embryonic lethality (Yamamoto et al., 2007). Analysis of
mouse embryonic fibroblasts (MEFs) deficient in either
ATF6α or ATF6β showed that ATF6α but not ATF6β
is required for transcriptional induction of not only ER
chaperones but also ERAD components, and that
ATF6α–/– MEFs are sensitive to ER stress. Wu et al. inde-
pendently generated and characterized ATF6α-knockout
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Y. Adachi et al.
mice as well as MEFs deficient in ATF6α, and reached
similar but not identical conclusions (Wu et al., 2007) (see
the Discussion for details).
Our previous analysis focused on selected canonical tar-
get genes of the UPR. Here, to unambiguously clarify the
role of ATF6α, we performed a genome-wide search for
ATF6α-target genes. Based on the results, we propose that
ATF6 is a transcription factor which is specialized in the
regulation of ER quality control proteins.
Experimental Procedures
Preparation, culture and transfection of ATF6α+/+ and
ATF6α–/– MEFs
Male heterozygotes of ATF6α (ATF6α+/–) (Yamamoto et
al., 2007) were backcrossed to female wild-type mice
(C57BL/6J) eight times to obtain ATF6α N8-heterozygotes.
Crosses between male and female ATF6α N8-heterozygotes
were dissected on embryonic day 13.5 and MEFs were iso-
lated by trypsinization of embryos. Primary N8-MEFs were
cultured in Dulbecco’s modified Eagle’s medium (glucose
at 4.5 g/liter) supplemented with 10% fetal bovine serum,
2 mM glutamine, and antibiotics (100 U/ml penicillin and
100 µg/ml streptomycin) at 37°C in a humidified 5% CO2/
95% air atmosphere. Transfection was performed using
FuGENE6 (Roche) according to the manufacturer’s instruc-
tions. pECFP-N1-tsVSVG and pECFP-N1-A1AT to express
tsVSVG-CFP and A1AT-CFP fusion proteins, respectively,
were as described previously (Nadanaka et al., 2004).
PERK+/+ and PERK–/– MEFs (Harding et al., 2000b) were
the generous gift of Dr. David Ron (New York University).
XBP1+/+ and XBP1–/– MEFs (Lee et al., 2003) were the
generous gift of Dr. Laurie Glimcher (Harvard Medical
School).
Microarray analysis
Total RNA extracted from ATF6α+/+ and ATF6α–/– N8-
MEFs by the acid guanidinium/phenol/chloroform method
using ISOGEN (Nippon Gene) was further purified using
RNeasy Mini (Qiagen), and checked for quality with an
RNA 6000 Nano Assay using an Agilent 2100 Bioanalyser
(Agilent Technologies). Five hundred nanogram aliquots of
total RNA prepared from N8-MEFs untreated or treated
with 2 µg/ml tunicamycin for 8 h were converted to cDNA
by reverse transcription. cDNA obtained from untreated and
tunicamycin-treated N8-MEFs was then labeled by tran-
scription with cyanine 3-CTP and cyanine 5-CTP, respec-
tively, using an Agilent Low Input Linear Amplification kit.
After purification through RNeasy Mini, 825 ng each of
the labeled cRNA probes was mixed and hybridized with a
4×44K Agilent oligo microarray (Whole Mouse Genome),
on which 44,000 mouse genes were spotted, using an
ATF6 as a Specialized Transcription Factor for ER Quality Control
Agilent Gene Expression Hybridization Kit. Cyanine 3 and
cyanine 5 fluorescence intensities of a spot were obtained
after subtraction of respective background intensity of the
spot using a GenePix 4000B (Axon). Fold induction caused
by tunicamycin treatment was defined as the ratio of
cyanine 5 intensity to cyanine 3 intensity. Because our anal-
ysis was based on fold induction values, genes showing
extremely low fluorescence intensity were eliminated to
ensure accuracy. To this end, background intensity obtained
for 44,000 spots was summed and the average background
intensity was determined for both cyanine 5 and cyanine 3:
if cyanine 5 or cyanine 3 intensity for a spot was less than
half the respective average background intensity, the fold
induction value was not determined. We carried out four
independent experiments and obtained fold induction values
for 14,729 of 44,000 genes at least three and mostly four
times.
Northern blot hybridization
Total RNA was extracted from cultured N8-MEFs using
ISOGEN. Northern blot hybridization was performed
according to standard procedures (Sambrook et al., 1989).
Digoxigenin (DIG)-labeled cDNA probes were prepared
using PCR according to the manufacturer’s instructions
(Roche) and hybridized with RNA electrophoresed and
blotted on a membrane. Subsequent reaction with anti-
digoxigenin antibody (Roche) and treatment with the
chemiluminescent detection reagent CDP-star (GE Health-
care Biosciences) were performed according to the manu-
facturer’s specifications. Chemiluminescence was visualized
using an LAS-3000mini LuminoImage analyzer (Fuji Film).
Immunological techniques
Immunoblotting analysis was carried out according to the
standard procedure (Sambrook et al., 1989) as described
previously (Okada et al., 2002) using Western Blotting
Luminol Reagent (Santa Cruz Biotechnology). Chemi-
luminescence was detected using an LAS-3000mini Lumino-
Image analyzer (Fuji Film). ATF6α was detected with
rabbit anti-ATF6α polyclonal antibody (Haze et al., 1999).
Anti-GFP monoclonal antibody (mixture of clone 7.1 and
13.1) was purchased from Roche. Mouse anti-KDEL anti-
body was purchased from Stressgen. Immunoprecipitation
was carried out essentially as described previously (Nadanaka
et al., 2004).
Results
Identification of ATF6α-target genes
Using total RNA isolated from ATF6α+/+ and ATF6α–/–
MEFs which had been untreated or treated for 8 h with
77
tunicamycin, an inhibitor of protein N-glycosylation known
to evoke ER stress (Kaufman, 1999), we conducted four
independent microarray analyses, and obtained fold-
induction values for 14,729 of 44,000 spotted mouse genes
at least three and mostly four times (see Experimental Proce-
dures). The number of genes induced more than 2-fold by
tunicamycin treatment was 680 and 556 in ATF6α+/+
and ATF6α–/– MEFs, respectively. ATF6α targets were
defined as genes whose fold induction value in ATF6α+/+
MEFs was more than 2 and in ATF6α–/– MEFs was less
than half that in ATF6α+/+ MEFs. This process produced a
total of 30 genes as ATF6α-target genes (Table I), which
were categorized into several functional groups as detailed
below.
Requirement of ATF6α for transcriptional induction of
most ER chaperones
Microarray analysis showed that mRNA encoding the major
ER chaperone BiP (Sitia and Braakman, 2003) was induced
11-fold in ATF6α+/+ MEFs in response to tunicamycin
treatment (Fig. 1A). This induction was mitigated to less than
5-fold in ATF6α–/– MEFs. Similarly, the transcriptional
induction of ER-localized molecular chaperones ORP150/
GRP170, GRP94 and calreticulin (Sitia and Braakman,
2003) observed in ATF6α+/+ MEFs was greatly mitigated
in ATF6α–/– MEFs (Fig. 1A). These results are consistent
with our previous northern blot hybridization analysis of
BiP as well as immunoblotting analysis of BiP and GRP94
(Yamamoto et al., 2007), demonstrating that ATF6α is gen-
erally required for transcriptional induction of ER-localized
molecular chaperones, with the possible exception of
calnexin (Sitia and Braakman, 2003) and STCH (Otterson
et al., 1994). Transcriptional induction of GRP75, a mitochon-
drial molecular chaperone (Szabadkai et al., 2006), did
not depend on ATF6α, as expected.
ERp72, P5 and GRP58 are protein disulfide isomerase-
like folding enzymes containing thioredoxin motifs (Sitia
and Braakman, 2003). As shown in Fig. 1B, transcriptional
induction of these three enzymes observed in ATF6α+/+
MEFs was lost or greatly mitigated in ATF6α–/– MEFs.
After oxidization of substrates, protein disulfide isomerase
is reoxidized by the action of Ero1, which consists of
closely related ERO1α and ERO1β in mammals (Sitia and
Braakman, 2003). Microarray analysis showed that weak
transcriptional induction of ERO1α occurred similarly in
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-
vations were well confirmed by northern blot hybridization
analysis of MEFs treated with tunicamycin or thapsigargin,
an inhibitor of ER-Ca2+-ATPase known to evoke ER stress
(Kaufman, 1999), as shown in Fig. 1C.
p58IPK (Rutkowski et al., 2007), ERdj3 (Shen and
Hendershot, 2005) and ERdj4 (Shen et al., 2002b) are DnaJ
 Y. Adachi et al.

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

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

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

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

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ATF6 as a Specialized Transcription Factor for ER Quality Control

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.

83

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
in ATF6α–/– MEFs as compared with ATF6α+/+ MEFs.
Further, transcriptional induction of several other proteins
involved in translocation was also not dependant on
ATF6α, although the extent of their induction was low.
Similarly, three proteins involved in the ER-Golgi trans-
port, namely SEC23b (Paccaud et al., 1996), VDP (Allan
et al., 2000) and BET1 (Zhang et al., 1997), were also
induced in ATF6α+/+ and ATF6α–/– MEFs, as determined
by microarray analysis (Fig. 5B) and confirmed by northern
blot hybridization analysis (Fig. 5C). Importantly, transcrip-
tional induction of SEC23b, VDP and BET1 as well as
SRP54 required XBP1 as their mRNA was induced in
XBP1+/+ MEFs but not in XBP1–/– MEFs (Fig. 6); BiP
mRNA as a control was induced similarly in XBP1+/+ and
XBP1–/– MEFs, as reported previously (Lee et al., 2003;
Lee et al., 2002). These results indicated that ATF6 is not
required for transcriptional induction of either translocation
proteins or transport proteins.
We finally examined whether the absence of ATF6α
affects the transport of cargo proteins from the ER to the
Golgi apparatus using a temperature-sensitive mutant of
vesicular stomatitis virus G protein (tsVSVG) as a model
protein. At the non-permissive temperature of 39.5°C,
tsVSVG is misfolded due to a point mutation in its luminal
domain and retained in the ER. After shift to the permissive
84
Y. Adachi et al.
temperature of 32°C, in contrast, it is rapidly folded and
then transported to the plasma membrane through the Golgi
apparatus (Nehls et al., 2000). As shown in Fig. 7A, after
temperature downshift to 32°C, tsVSVG fusing to cyan-
emitting green fluorescent protein (tsVSVG-CFP) showed
doublet protein bands (upper panel), which were resolved
more clearly when samples were treated with endo-
glycosidase H (lower panel). As the endoglycosidase H-
resistant form (upper migrating band) represents the protein
modified at the Golgi apparatus, tsVSVG-CFP moved to
the Golgi apparatus with similar kinetics in ATF6α+/+
and ATF6α–/– MEFs.
We also examined whether secretion of α1-antitrypsin
(A1AT), a serum glycoprotein, was affected by the absence
of ATF6α. We expressed A1AT-CFP fusion protein by
transfection and found that A1AT-CFP was secreted simi-
larly in ATF6α+/+ and ATF6α–/– MEFs even in the pres-
ence of 1 mM dithiothreitol, a reducing reagent known to
cause ER stress (Kaufman, 1999), as shown in Fig. 7B: as
A1AT contains only one cysteine residue, dithiothreitol
should have no effect on the folding of A1AT (Nadanaka et
al., 2004). In ATF6α+/+ or ATF6α–/– MEFs treated with
tunicamycin, in contrast, A1AT was not secreted and its
deglycosylated and thus misfolded form was accumulated
intracellularly (Fig. 7B). These results indicated that cargo
proteins are transported from the ER normally in ATF6α–/–
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-
port from the ER to the Golgi apparatus was not affected by
the absence of ATF6α.
Discussion
A prototype of the UPR was first discovered in the 1970s,
when analysis of virus-transformed mammalian cells identi-
fied GRP78 and GRP94 as proteins inducible by glucose
starvation (Shiu et al., 1977). Substantial analysis of
the UPR began in the late 1980s, when the accumulation
of unfolded proteins in the ER was found to trigger the
induction of GRP78 (found to be identical to the major ER
chaperone BiP) and GRP94 (found to be a molecular
chaperone of the Hsp90 family) (Kozutsumi et al., 1988).
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
unfolded proteins. The use of the budding yeast Saccharomyces
cerevisiae as a model in the 1990s led to major progress in
understanding the molecular mechanisms of the UPR, and
to molecular cloning of the UPR transducer Ire1p present in
the ER (Cox et al., 1993; Mori et al., 1993), as well as the
UPR-specific transcription factor Hac1p (Cox and Walter,
1996; Mori et al., 1996); followed by discovery of Ire1p-
mediated splicing of HAC1 mRNA, which connects the event
in the ER to that in the nucleus (Cox and Walter, 1996;

85

Kawahara et al., 1997). A number of ER chaperones were
identified as targets of the Ire1p-Hac1p pathway in the yeast
UPR (Mori et al., 1998). Importantly, microarray analysis, a
new technology invented in the late 1990s, increased the
number of UPR target genes drastically, to 381 out of a total
6,000 yeast genes, including not only ER chaperones but
also numerous proteins working at various stages of secre-
tion, specifically proteins involved in translocation, protein
folding, protein degradation, glycosylation in the ER,
lipid/inositol metabolism, ER-Golgi transport, Golgi-ER
retrieval, glycosylation in the Golgi apparatus, vacuolar tar-
geting, distal secretion and cell wall biogenesis. Based on
these findings, it was proposed that activation of the yeast
UPR induces specific remodeling of the secretory pathway
to minimize the amount, concentration, or both of unfolded
proteins in the ER (Travers et al., 2000).
In metazoan cells, the UPR transducer Ire1p is well con-
served, whereas the target transcription factor of Ire1p-
mediated unconventional splicing is switched from Hac1p
to XBP1 (Calfon et al., 2002; Shen et al., 2001; Yoshida
et al., 2001a). The IRE1-XBP1 pathway is responsible for in-
duction of most canonical UPR target genes in C. elegance
(Shen et al., 2005) and D. melanogaster (Hollien and
Weissman, 2006). Interestingly, however, the IRE1-XBP1
pathway is dispensable to the induction of major ER chaper-
ones such as GRP78/BiP and GRP94 in mammalian cells,
but is required for induction of a subset of ER chaperones
and most ERAD components (Lee et al., 2003; Lee et al.,
2002; Oda et al., 2006; Yoshida et al., 2003), thus revealing
diversity in the transcriptional induction system in mam-
mals. Extensive genome-wide searches using microarray or
chromatin immunoprecipitation analyses by several labo-
ratories consistently showed that mammalian XBP1 is
involved in the expression of not only genes working for
protein folding and degradation in the ER but also many
genes working for the secretory pathway and others, simi-
larly to the case of S. cerevisiae, indicating that XBP1 is a
multifunctional transcription factor (Acosta-Alvear et al.,
2007; Lee et al., 2003; Shaffer et al., 2004; Sriburi et al.,
2007).
The UPR transducer ATF6 is present in metazoan
genomes but not in the yeast genome. Worm and fly
genomes contain a single ATF6 gene, whereas mammalian
genomes contain two closely related genes, ATF6α and
ATF6β. Although worm and fly ATF6 has little role in the
UPR (Hollien and Weissman, 2006; Shen et al., 2005), we
recently showed that mammalian ATF6α but not ATF6β
is required for transcriptional induction of major ER
chaperones as well as of ERAD components. Detailed
analysis showed that ATF6α is solely responsible for the
transcriptional induction of major ER chaperones and that
ATF6α heterodimerizes with XBP1 for the induction of
major ERAD components (Yamamoto et al., 2007). Thus,
ATF6 has gained the ability to induce the canonical UPR
target genes in higher eukaryotes.
86
Y. Adachi et al.
In the present study, we conducted a genome-wide search
for ATF6α-target genes by microarray analysis of MEFs
deficient in ATF6α. Among the 14,729 mouse genes we
could successfully analyze, 680 and 556 genes were
induced more than 2-fold in response to tunicamycin treat-
ment of ATF6α+/+ and ATF6α–/– MEFs, respectively.
When the following criteria were applied, however, only 30
genes were identified as ATF6α-target genes: the ATF6α-
target gene had to be induced more than 2-fold in ATF6α+/+
MEFs and its fold induction value in ATF6α–/– MEFs had
to be less than half that in ATF6α+/+ MEFs. Among these
30 genes, notably, 7 were ER chaperones, 5 were ERAD
components and 6 were ER proteins; six ER proteins are
SDF2L1 for protein O-glycosylation (Fukuda et al., 2001);
CRELD2 with unknown function (Ortiz et al., 2005);
ARMET with unknown function (Mizobuchi et al., 2007);
PIG-A for glycosylphosphatidylinositol anchor biosynthesis
(Watanabe et al., 1996); SERCA2, Ca2+-ATPase (Thuerauf
et al., 2001); and ORMDL2 for protection from ER stress
(Hjelmqvist et al., 2002) (Table I). The remaining 12 targets
were miscellaneous and could not be further categorized.
ATF6α therefore appears to have limited function as com-
pared with XBP1, and may regulate ER quality control pro-
teins primarily and specifically.
It is particularly noteworthy that proteins involved in
translocation into the ER (SRP54, SSR3 and SPCS3) as
well as ER-Golgi transport (SEC23b, VDP and BET1) are
not ATF6α targets, but rather XBP1-targets (Figs. 5 and 6).
We did not observe any defects in the transport of cargo
proteins (tsVSVG and A1AT) out of the ER to the plasma
membrane or extracellular space via the Golgi apparatus in
ATF6α–/– MEFs as compared with ATF6α+/+ MEFs (Fig.
7). Wu et al. independently generated ATF6α-knockout
mice and characterized ATF6α-deficient MEFs (Wu et al.,
2007). Their conclusions were similar to ours as far as regu-
lation of ER chaperones and ERAD components are con-
cerned. Nonetheless, they found that transport of transferin
receptor from the ER to the Golgi apparatus or secretion of
alkaline phosphatase into the extracellular space was ineffi-
cient in ATF6α–/– MEFs treated with thapsigargin as com-
pared with untreated ATF6α–/– MEFs or ATF6α+/+ MEFs
treated with thapsigargin, leading them to propose that
ATF6α is required to optimize protein folding, secretion
and degradation during ER stress. In their microarray analy-
sis, however, only Rab38 was identified as an ATF6α-target
among numerous proteins involved in vesicular transport if
our criteria for ATF6α-target genes were applied, and Rab38
appeared to control post-Golgi trafficking (Wasmeier et al.,
2006). In our analysis, signals of Rab38 were extremely
weak and thus Rab38 was excluded from the 14,729 genes
we analyzed. Therefore, the molecular basis of their pro-
posal regarding secretion defects of ATF6α–/– MEFs seems
unsupported. Rather, we suspect that the inefficient trans-
port of transferin receptor or alkaline phosphatase in
ATF6α–/– MEFs treated with thapsigargin as compared
ATF6 as a Specialized Transcription Factor for ER Quality Control
with similarly treated ATF6α+/+ MEFs may be due to fold-
ing defects resulting from inefficient induction of ER
chaperones. It is markedly difficult to separate transport
defects from folding defects experimentally, because only
correctly folded cargo proteins are transported. In our
experiments with tsVSVG (Fig. 7A), transfected cells were
incubated at the non-permissive temperature 39.5°C for
18 h to cause misfolding and retention of tsVSVG in the
ER, which should have evoked significant ER stress in both
ATF6α+/+ and ATF6α–/– MEFs. Yet, tsVSVG was trans-
ported to the Golgi apparatus similarly in ATF6α+/+ and
ATF6α–/– MEFs after shift to the permissive temperature
32°C. We therefore concluded that ER-Golgi transport is
not regulated by ATF6α and that ATF6α is a transcription
factor which is specialized in regulating the expression of
ER quality control proteins.
Based on the present and previously published results, we
envision the following scenario to explain the evolution of
the UPR. First, the IRE1 pathway evolved to counteract the
accumulation of unfolded proteins in the ER. Because cells
such as S. cerevisiae keep synthesizing proteins even under
ER stress conditions at this evolutional stage, activated
IRE1 induces transcription of not only ER chaperones and
ERAD components but also numerous proteins working at
various stages of secretion to minimize the amount, concen-
tration or both of unfolded proteins accumulated in the ER
(Travers et al., 2000). Next, the PERK pathway emerged
via gene shuffling between IRE1 and GCN2: GCN2
encodes a protein kinase which phosphorylates eIF2α in
response to amino acid starvation (Silverman and Williams,
1999). Cell such as C. elegance would now be able to atten-
uate translation and decrease the burden on the ER, which
was perhaps advantageous in handling unfolded proteins in
multicellular organisms, as these encounter not only envi-
ronmental but also physiological ER stress during develop-
ment or differentiation. The ATF6 pathway then evolved,
which is specialized in regulating the transcription of ER
quality control proteins. Because of the difference between
the IRE1 and ATF6 pathways in their mechanisms of acti-
vating downstream transcription factors, cells such as those
in mammals are able to perform a time-dependent phase
shift to cope with ER stress in accordance with the quality,
quantity, or both of unfolded proteins accumulated in the
ER (Yoshida et al., 2003). Finally, various tissue-specific
UPR transducers, such as IRE1β (Bertolotti et al., 2001)
and ATF6-like membrane-bound transcription factors, i.e.
OASIS (Kondo et al., 2005), CREBH (Zhang et al., 2006),
Luman/LZIP (Raggo et al., 2002) and Tisp40 (Nagamori et
al., 2005), were developed to strengthen local quality con-
trol capability in the ER. It appears that ER stress is so
threatening to the cell that ever more sophisticated tactics
were created in accordance with the complexity of biologi-
cal systems in the cell or organism constructed during evo-
lution.
87
Acknowledgements. We are grateful to Dr. D. Ron for his provision of
PERK+/+ and PERK–/– MEFs and Dr. L. Glimcher for XBP1+/+ and
XBP1–/– MEFs. We thank Ms. Kaoru Miyagawa for her technical and
secretarial assistance. This work was supported in part by grants from the
Ministry of Education, Culture, Sports, Science and Technology of Japan
(15GS0310 and 19058009 to K. M.).
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(Received for publication, December 25, 2007
and accepted, January 31, 2008)