1. In vitro reconstitution of ER-stress induced ATF6
transport in COPII vesicles
Adam J. Schindler and Randy Schekman1
Department of Molecular and Cell Biology and Howard Hughes Medical Institute, Barker Hall, University of California, Berkeley, CA 94720-3202
Contributed by Randy Schekman, September 9, 2009 (sent for review July 27, 2009)
The transcription factor ATF6 is held as a membrane precursor in
the endoplasmic reticulum (ER), and is transported and proteolyti-
cally processed in the Golgi apparatus under conditions of un-
folded protein response stress. We show that during stress, ATF6
forms an interaction with COPII, the protein complex required for
vesicular traffic of cargo proteins from the ER. Using an in vitro
budding reaction that recapitulates the ER-stress induced transport
of ATF6, we show that no cytoplasmic proteins other than COPII are
necessary for transport. ATF6 is retained in the ER by association
with the chaperone BiP (GRP78). In the in vitro reaction, the
ATF6-BiP complex disassembles when membranes are treated with
reducing agent and ATP. A hybrid protein with the ATF6 cytoplas-
mic domain replaced by a constitutive sorting signal (Sec22b
SNARE) retains stress-responsive transport in vivo and in vitro.
These results suggest that unfolded proteins or an ER luminal ؊SH
reactive bond controls BiP-ATF6 stability and access of ATF6 to the
COPII budding machinery.
regulated transport ͉ unfolded protein response ͉ BiP ͉
prebudding complex
Endoplasmic reticulum (ER) homeostasis can be perturbed by
up-regulation of secretory proteins or by disruption of
protein processing, a condition termed ER stress (1). ER stress
causes increased protein misfolding and leads to activation of the
unfolded protein response (UPR), an evolutionarily conserved
pathway to alleviate ER stress (2). The UPR leads to a slowdown
in protein synthesis, an upregulation of ER chaperones, and an
upregulation of ER-associated protein degradation.
The three effector proteins for the UPR are IRE1, PERK, and
ATF6. During the UPR, IRE and PERK remain in the ER and
act via cytoplasmic effectors, whereas ATF6 is transported to the
Golgi complex. In the Golgi, ATF6 is cleaved by Site-1 and Site-2
proteases (3). These cleavages release the N-terminal cytoplas-
mic domain, which contains a bZIP motif that binds DNA at ER
stress response elements that control expression of the ER
chaperones BiP (GRP78) and GRP94 (4).
Transport of ATF6 and other cargo proteins likely involves the
COPII coat, a complex of five cytoplasmic proteins that selects
cargo at the ER membrane and pinches off membranes to form
vesicles. The five proteins are the GTPase Sar1, which is
recruited to the ER membrane and initiates coat formation by
GDP to GTP exchange; the heterodimeric complex Sec23/Sec24,
which binds to Sar1⅐GTP; and a second dimeric complex,
Sec13/Sec31, which binds to Sec23/24 and provides the curvature
necessary for vesicle fission (5). Cargo is selected by interactions
between domains on the Sec24 subunit and cytoplasmic motifs
on cargo proteins (6). Inhibition of COPII by overexpression of
dominant negative Sar1 has been shown to block transport of
ATF6 (7).
A central question in ATF6 function is how luminal stress is
converted into recognition by the cytoplasmic COPII complex.
The luminal chaperone BiP binds ATF6 stably in unstressed cells
and dissociates specifically during stress (8). The triggers for this
release are unclear. Structural studies of IRE1, which is also
controlled by BiP, have posited direct recognition of misfolded
proteins that induces a conformation change to the active state
(9, 10). Whether ATF6 is triggered by a similar mechanism of
misfolded protein binding is unclear.
Evidence supports a model in which accessory proteins are
necessary for ATF6 transport. When the luminal domain of
ATF6 was fused to the cytoplasmic domain of the ER-localized
protein LZIP, the chimera still trafficked to the Golgi during ER
stress (11), demonstrating that the ATF6 cytoplasmic domain is
not required for transport. A sorting signal may be provided by
a cargo receptor that engages ATF6 in the ER lumen and COPII
in the cytoplasm, an interaction occurring specifically during
stress.
We have established in vitro reactions that recapitulate ATF6
dissociation from BiP and engagement with COPII during stress.
These data show that during stress ATF6 undergoes alterations
that deliver it to COPII vesicles for transport.
Results
ATF6 Transport Is Recapitulated in Vitro in a Cell Line Stably Express-
ing FLAG-ATF6. To assess ATF6 transport, we generated a stable
CHO cell line, CHO-ATF6, expressing full-length ATF6 with
three copies of the FLAG epitope at the N terminus (12).
CHO-ATF6 cells were used to measure the response of ATF6 to
ER stress (Fig. 1A). Full-length ATF6 at 110 kDa represents the
ER-localized protein, and the band at 65 kDa represents ATF6
that has trafficked to the Golgi and been cleaved. Cells were
treated with the ER stress inducers dithiothreitol (DTT), tuni-
camycin (Tm), or thapsigargin (Tg) for varying times. DTT
breaks disulfide bonds to unfold proteins, Tm blocks N-
glycosylation of nascent polypeptides, and Tg inhibits ER cal-
cium pumps. As a control, cells were treated with hydrogen
peroxide (H2O2), an oxidative stress agent. DTT induced a rapid
shift in ATF6 to the processed form, causing cleavage of greater
than half of cellular ATF6 within 30 min. Tm and Tg were both
slower-acting, requiring 2–4 h for maximum effect. H2O2 did
not cause ATF6 cleavage (Fig. 1A). The localization of ATF6
was examined by immunofluorescence. ATF6 in unstressed
or H2O2-treated cells was diffuse in the ER. During stress,
ATF6 partially localized with a Golgi protein, Gos28, and
displayed a more prominent nuclear localization, consistent with
its trafficking (Fig. S1).
To assess the mechanisms of ATF6 transport, we used an in
vitro assay that recapitulated ER stress-induced vesicle budding.
CHO-ATF6 cells were permeabilized with digitonin to allow
access to intracellular organelles. This technique has been shown
to generate functional ER membranes that maintain protein
topology and resist protease treatment (13, 14). In vitro COPII
vesicle budding from the ER was induced by addition of GTP,
rat liver cytosol, ATP, and an ATP regeneration system. After
incubation to allow vesicle formation, membranes were removed
Author contributions: A.J.S. and R.S. designed research; A.J.S. performed research; A.J.S.
contributed new reagents/analytic tools; A.J.S. and R.S. analyzed data; and A.J.S. and R.S.
wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: schekman@berkeley.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0910342106/DCSupplemental.
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CELLBIOLOGY

2. by sedimentation at low speed, and budded vesicles were collected
by sedimentation at high speed. The packaging of 3ϫ-FLAG-ATF6
was compared with a positive control protein, ERGIC-53, a
lectin that cycles between the ER and ER-Golgi intermediate
compartment, and a negative control, Ribophorin-I, part of the
oligosaccharyl transferase complex and a resident ER protein.
As a second positive control, we examined the budding of
Sec22b, a v-SNARE that transports constitutively between the
ER and Golgi.
ATF6 budded poorly in the standard reaction (Fig. 1B, lanes 4
and 5). When DTT was added into the reaction, ATF6 was enriched
in vesicles (lanes 6–8). DTT did not affect ERGIC-53 or Sec22b
budding, and did not cause significant Ribophorin-I release. No
other toxin caused ATF6 budding. The inability of Tm and Tg to
package ATF6 may seem in conflict with data from the cleavage
assay (Fig. 1A). However, DTT can act on folded proteins directly
by reducing disulfide bonds, whereas Tm and Tg may act only
indirectly on proteins as they are synthesized, conditions that likely
do not occur in the cell-free transport reaction. Other reports have
also shown that DTT, but not Tg, can induce ATF6 transport in the
absence of protein synthesis (15).
ATF6 packaging in the presence of DTT was 2- to 5-fold
higher than in untreated membranes. The overall efficiency of
ATF6 packaging in the presence of DTT was Ϸ5–10%. The
efficiency of ATF6 transport was lower in vitro than in vivo after
1 h treatment with DTT. The decreased efficiency may be
accounted for by the inaccessibility of the majority of natively
folded, disulfide-linked proteins to reducing agents (16). DTT
will likely generate higher levels of unfolded proteins in cells
engaged in protein synthesis, conditions that are not reproduced
in our cell-free reaction.
We next examined the budding of endogenous ATF6 to
determine whether the cell-free reaction reproduced transport
of physiological levels of ATF6 (Fig. 1C). We found that
endogenous ATF6 in CHO.K1 cells was packaged with similar
efficiency in response to DTT as FLAG-tagged ATF6 in stably
transfected cells, demonstrating that the effect is valid for ATF6
expressed at normal levels.
In Vitro Packaging of ATF6 Requires Luminal Reducing Activity and
COPII Proteins. We probed the requirement for a reducing agent
using permeabilized cells treated with two membrane-
permeable reducing agents, DTT and ␤-mercaptoethanol
(BME), and a membrane-impermeable reducing agent, Tris(2-
Carboxyethyl) phosphine hydrochloride (TCEP) (Fig. 2A) (17).
Both DTT and BME stimulated packaging, whereas TCEP had
a small stimulatory effect at lower concentration and an inhib-
itory effect at higher concentration. The reducing potential of
TCEP is greater than that of DTT at neutral pH (18); therefore,
the levels of all chemicals were comparable. These results show
that reducing activity in the lumen of the ER is necessary for
ATF6 packaging into vesicles.
Fig. 1. ATF6 transports in vivo and in vitro in stably expressing cells. (A) CHO.K1 cells stably expressing 3ϫ-FLAG-ATF6 were treated in 24-well culture dishes
with toxins for indicated times. Full-length protein is 110 kDa, cleaved protein is 65 kDa. (B) Permeabilized CHO-ATF6 cells were incubated with GTP, ATP, and
an ATP-regenerating system (ATPr), and 4 mg/mL rat liver cytosol for 1 h at room temperatur. Lane 1, 20% load of starting membranes; lanes 2 and 3, controls,
in which either nucleotides or rat liver cytosol were excluded; lanes 4 and 5, duplicate incubations of the full budding reaction; lanes 6–17, full reactions with
the addition of indicated toxins. Control proteins analyzed in parallel with ATF6 were ERGIC-53, a constitutively transported membrane lectin; Sec22b, a
constitutively transported v-SNARE, and Ribophorin-I, a component of the oligosaccharyl transferase complex and resident ER protein. Proteins were analyzed
by SDS/PAGE and immunoblotting. Quantification was performed by comparing the signal in vesicle lanes to the 20% starting membrane signal in lane 1. (C)
Budding reaction in CHO.K1 cells with immunoblotting to endogenous ATF6. *, Nonspecific band.
Fig. 2. In vitro budding reaction recapitulates ER-stress induced ATF6 trans-
port. (A) Budding reaction in the presence of membrane permeable reducing
agents DTT and BME, or membrane-impermeable reducing agent TCEP.
(B) Budding reactions conducted in the presence of COPII inhibitors GTP␥S
and Sar1H79G, or controls GDP and Sar1, or in the presence of the COPI
inhibitor Brefeldin A or the proteasome inhibitor ALLN. All lanes contained
2 mM DTT.
17776 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910342106 Schindler and Schekman

3. Selective inhibitors were used to assess the role of COPII in
ATF6 budding. A nonhydrolyzable GTP analog, GTP␥S, and a
dominant negative Sar1, Sar1H79G, both blocked budding of
ATF6 and ERGIC-53 (Fig. 2B). GDP had no effect, and WT
Sar1 enhanced budding. Brefeldin A, which prevents retrograde
transport from Golgi to ER, had a moderately inhibitory effect
on all cargo, and the proteasome inhibitor ALLN had no effect.
We conclude that ATF6 packaging depends on COPII.
Budded vesicles convey ATF6 to the Golgi membrane in vivo.
Fusion of vesicles at the Golgi exposes ATF6 to Site-1 and Site-2
protease processing. We examined whether this fusion event
occurred in our in vitro assay. We found no processing of ATF6,
suggesting that the full transport event was not sustained in this
reaction (Fig. S2).
ATF6 Physically Interacts with COPII for Transport. We examined the
physical interaction of ATF6 with COPII on ER membranes
using a prebudding assay in which purified COPII proteins are
recruited to membranes and COPII-bound ER proteins isolated.
The Sec23/24 complex is hypothesized to bind to a GTP-
restricted Sar1 mutant, Sar1H79G, but not to a GDP-restricted
Sar1, Sar1T39N (Fig. 3A). Cargo is captured by Sec23/24 on
membranes.
In the experiment, GST-Sar1 and Sec23/24 were mixed with
membranes and the bound proteins on sedimented membranes
solubilized with detergent to permit isolation of cargo complexes
by immobilized glutathione. Salt-washed, permeabilized cells
were untreated or pretreated with 2 mM DTT for 15 min in
culture to mobilize ATF6 to ER exit sites. Immunoblot results
(Fig. 3B) showed that both forms of GST-Sar1 were recruited to
membranes, with Sar1T39N binding slightly better to mem-
branes than Sar1H7G (Fig. 4B, ‘‘membrane bound’’ lanes 3 and
4, and 7 and 8). Sec23 bound to membranes incubated with
Sar1H79G, but less well to membranes incubated with Sar1T39N,
consistent with the requirement for GTP (lanes 1 and 2, and 5
and 6). ERGIC-53 served as a control protein for specific cargo
binding, and Ribophorin-I served as a control for nonspecific
binding. ERGIC-53 was recruited to Sar1H79G/Sec23/24, whereas
Fig. 3. ATF6 forms a physical association with COPII proteins and transports without additional cytoplasmic proteins. (A) Diagram of the prebudding assay
scheme to detect associations between ATF6 and COPII. (Left) GDP-restricted form of Sar1, Sar1T39N, is not able to recruit the Sec23/24 complex to membranes,
and cargo proteins (such as ATF6) are not precipitated by glutathione pulldown to Sar1. (Right) GTP-restricted Sar1, Sar1H79G, recruits Sec23/24 and cargo
proteins. ATF6 binding to COPII complexes is hypothesized to require DTT stimulation. Adapted from (25). (B) Prebudding assay in the presence of 10 ␮g of the
Sec23/24 complex and 5 ␮g of GST-Sar1. (Left) Absence of 2 mM DTT pretreatment; (Right) After DTT pretreatment. Lanes 1 and 2, and 7 and 8 contain
supernatants that did not bind to membranes in the reaction. These lanes demonstrate that Sec23/24 preferentially binds to Sar1⅐GTP, because unbound Sec23/24
is elevated in the presence of Sar1T39N and depleted in the presence of Sar1H79G. Lanes 3 and 4, and 9 and 10 contain proteins bound to membranes and solubilized
by detergent. These lanes contain resident ER proteins and soluble proteins that adhered to membranes. Lane 5 and 6, and 11 and 12 are proteins extracted after
glutathione pulldown of GST-Sar1 complexes. Cargo proteins ERGIC-53 and ATF6 are elevated in the Sar1H79G condition. ATF6 is present specifically after
pretreatment with DTT (arrow). Bound fractions were 30% of the total; lysates were 1% of the total. (C) Quantification of four assays as in B. Binding efficiency
was determined by subtracting the T39N signal from the H79G signal in the beads lanes, and calculating the bound amount as a percentage of total protein in
lysates. n ϭ 4; *, P Ͻ 0.05. (D) Budding reaction in HeLa-ATF6 cells in the presence of COPII and absence of cytosol. First four lanes represent the standard budding
assay with 4 mg/mL cytosol. Remaining lanes had cytosol excluded and COPII added. Values for COPII indicate amounts each of Sar1, Sec23/24 complex, and
Sec13/31 complex.
Fig. 4. ATP and DTT act synergistically to dissociate BiP from ATF6. (A)
Immunoprecipitation of FLAG-ATF6 in permeabilized CHO-ATF6 cells incu-
bated for 30 min in the presence of 1 mM ATP or ATP␥S, or 5 mM DTT. After
incubation, cells were washed, lysed, and immunoprecipitated by ␣-FLAG
followed by immunoblotting to FLAG and BiP. (B) Quantification of two
replicates of assay as in A. BiP:ATF6 ratios were determined for individual
reactions and normalized to the value for untreated cells. (C) Budding reaction
to detect the presence of BiP in vesicles.
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4. Ribophorin-I was not (lanes 5 and 6, and 11 and 12). ATF6 was
recruited in the Sar1H79G/Sec23/24 condition after DTT treat-
ment (lane 12). In four assays, DTT-stimulated binding of ATF6
to COPII was detected at 3-fold above untreated samples (Fig.
3C). The efficiency of ATF6 binding was low compared with
ERGIC-53 (0.5% ATF6; 2.5% ERGIC-53), a difference com-
parable with that seen in the budding assay. These results
demonstrate that ATF6 forms physical association with COPII
proteins under conditions of ER stress.
We wished to assess the role of COPII as the sole cytosolic
requirement for ATF6 packaging in our cell-free reaction. Cells
were washed with 1 M KoAc to remove peripheral proteins, and
purified COPII was added in a titration experiment. ATF6 and
other cargo proteins were packaged in a COPII-concentration
dependent manner (Fig. 3C). ATF6, although less efficiently
packaged than other cargo, was most stimulated by increasing
amounts of COPII. These data demonstrate that ATF6 pack-
aging requires no cytosolic components other than COPII.
ATP and DTT Synergistically Induce BiP Dissociation from ATF6 in Vitro.
ATF6 dissociation from BiP is necessary for transport (12). To
test whether stress reproduces this effect in vitro, permeabilized
cells were treated with combinations of ATP and DTT, followed
by FLAG-ATF6 immunoprecipitation and immunoblot of BiP
(Fig. 4A). Treatment with ATP or DTT alone caused approx-
imately a 30% reduction in the ratio of BiP:ATF6 signal relative
to untreated cells (Fig. 4B). Although BiP has been found to
stably associate with ATF6 (8), the high levels of ATP in the
assay may allow some release of BiP. The combination of ATP
and DTT reduced the association to Ͻ50%, and ATP␥S, a
nonhydrolyzable analog of ATP, reduced the complex to Ͻ30%
of the untreated control. These data demonstrate that DTT and
ATP act together to release BiP from ATF6. This dissociation
may trigger access of ATF6 to COPII.
BiP is normally retained in the ER. We examined the effect
of DTT on the retention or transport of BiP (Fig. 4C). BiP was
found only at low levels in vesicles, and its packaging was not
induced by DTT. Thus, it appears that ATF6 is packaged in the
absence of BiP.
ATF6 Is Primed to Transport After in Vivo Treatment. We took
advantage of the ability of DTT to mobilize ATF6 rapidly in vivo
to assess whether DTT was required in the in vitro assay.
CHO-ATF6 cells were pretreated in culture for 15 or 60 min with
2 mM DTT. This short time period was designed to mobilize
ATF6 for transport while maintaining it in the ER. Permeabil-
ized cells were washed to remove DTT and incubated in the
budding reaction in parallel with nonpretreated controls (Fig.
5A). ATF6 packaging was enhanced after pretreatment even in
the absence of added DTT. In contrast, cells that were not
pretreated displayed the standard DTT sensitivity, with little
constitutive activity. A similar effect was seen after 60 min
treatment, although budding efficiency was reduced from 15
min. Thus, the COPII-ATF6 interaction in vitro does not require
the presence of reducing agent if ATF6 is primed for transport.
Treatment of membranes with high salt removes peripherally
attached complexes. We examined the effect of such a treatment
on DTT-induced packaging of ATF6 in vitro. Cells were either
untreated or pretreated for 15 min with DTT before harvest, and
both sets of permeabilized cells were salt washed with 1 M KoAc
for 15 min (Fig. 5B). This treatment reduced the overall budding
efficiency, but did not alter the effects of pretreatment seen in
Fig. 5A. These results demonstrate that the ATF6 complex
formed during ER stress is stable to high salt treatment that
removes most peripheral proteins.
ATF6 Is Actively Retained in the ER via Its Luminal Domain. It has been
established that the luminal domain of ATF6 controls its
localization independent of the cytoplasmic domain (12). We
examined the contribution of the ATF6 luminal domain in the
context of a constitutively transported protein, the v-SNARE
Sec22b. Both Sec22b and ATF6 are single-pass, type II mem-
brane proteins. The Sec22b sorting signal for COPII binding
has been mapped to a structural motif near the ER membrane
(19).
We conducted cleavage assays on ATF6 and on constructs in
which the ATF6 cytoplasmic domain was replaced with that of
Sec22b. A constitutive transport mutant, ATF6 1-500 (⌬431-475)
(12), was cleaved in the presence or absence of ER stress,
whereas a transport-deficient mutant ATF6 1-670 (⌬468-500)
(12), missing the luminal transport signal, remained intact with
or without DTT (Fig. 6A). These same constructs were gener-
ated with the Sec22b cytoplasmic domain (Fig. 6B) and also
examined by cleavage assay. Sec22b-ATF6TM migrated at the
predicted 22-kDa size (lanes 9 and 10). The chimeric protein
containing the full ATF6 luminal domain generated a cleaved
species predominantly in the presence of DTT (lanes 3 and 4).
The luminal domain mutants (lanes 5–8) displayed the same
phenotype as seen for ATF6 mutants (Fig. 6C).
We used the COPII budding reaction to examine Sec22b-
ATF6TM and Sec22b-ATF6 transport activity. The transmem-
brane-only construct budded in the absence or presence of DTT,
analogous to wild-type Sec22b. Sec22b-ATF6 transport showed
high constitutive transport, likely because the Sec22b domain
conferred some degree of transport. Still, it was stimulated
2.4-fold by DTT, showing that it was restrained in the absence
of stress. Thus, in cells and in the cell-free budding reaction, the
ATF6 retention signal overrode at least in part the constitutive
Sec22b sorting signal.
Discussion
We report a cell-free vesicle budding reaction that recapitulates
the ER-stress induced transport of the transcription factor
ATF6. The addition of a reducing agent, DTT, mobilized ATF6
into COPII vesicles from the ER in the absence of protein
synthesis. No other cytosolic adaptor proteins are required to
package ATF6 into COPII vesicles.
Fig. 5. Pretreatment in culture overcomes the requirement for DTT in vitro.
(A) CHO-ATF6 cells were pretreated in culture with 2 mM DTT for indicated
times and washed to remove DTT. Budding reactions were conducted in the
absence or presence of DTT. (B) Cells were pretreated as in A, permeabilized
and salt washed with 1 M KoAc for 15 min to remove peripheral membrane
proteins, followed by the budding reaction.
17778 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910342106 Schindler and Schekman

5. ATF6 dissociates from the luminal chaperon BiP (GRP78) in
response to exogenous reducing agent in cells and in our cell-free
reaction. The trigger for BiP dissociation may be proteins unfolded
in response to reducing agent or a specific ϪSH reactive sensor
protein, either of which may expose BiP to ATP binding and release
from ATF6. By analogy, antibody light chain binding to heavy chain
causes ATP release of BiP from heavy chain (20).
We observed a basal level of constitutive transport of ATF6 in
the absence of DTT, whereas the control protein Ribophorin-I
was entirely excluded. ATF6 may have low levels of basal
transport that serve to maintain cellular homeostasis. Reports
have shown a role for ATF6 in development (21) and in the
survival of dormant tumor cells (22).
The experiments with Sec22-ATF6 fusion proteins show that
ATF6 does not transport constitutively in the context of a strong
COPII sorting signal (19). ATF6 is present in a complex that
displays slow diffusional mobility in the ER, possibly suggesting
a higher-order complex (8). ATF6 may interact with additional
proteins besides BiP that restrain it from transport. Alterna-
tively, the slow diffusion of ATF6 in the ER under unstressed
conditions may limit its accessibility to COPII at ER exit sites.
These data suggest that BiP release from ATF6, triggered by
unfolded proteins or a stress-sensor protein, is followed by
recognition of ATF6 or a partner protein by COPII. Past
experiments showed that ATF6 still transported when its cyto-
plasmic domain was replaced with the cytoplasmic domain of an
ER-resident protein (11), suggesting that transport involves a
protein that mediates association between the luminal domain of
ATF6 and the cytoplasmic COPII proteins. There are many
examples of such receptors required to convey a membrane
cargo protein into COPII vesicles (23). Some receptors accom-
pany membrane cargo proteins into the vesicle, whereas others
are retained in the ER (24). In the case of regulated transport,
the best understood example is sterol control of SREBP, which
depends on COPII binding to the cargo receptor SCAP (25). A
proteomic analysis of COPII vesicles formed under conditions
permissive for ATF6 packaging may facilitate the identification
of its sorting receptor.
Methods
Plasmids and Cell Lines. The WT, ⌬431-475, and ⌬468-500 human ATF6␣ cDNA
with a 3ϫ N-terminal FLAG tag were gifts of R. Prywes (Columbia University,
New York, NY) and are described elsewhere (12). The original Sec22b cDNA
was a gift of J. Hay (University of Montana, Missoula, MT). Chimeric Sec22-
ATF6 proteins were made by amplifying Sec22b to include a 5Ј FLAG tag and
exclude the 3Ј transmembrane region. PCR fragments were cloned into the
pIRES2.neo (Clontech) backbone at StuI and ClaI sites. Transmembrane and
luminal regions of ATF6 were PCR amplified to include a 5Ј ClaI site and a 3Ј
XbaI site, and were cloned into the Sec22b.pIRES2 vector. All plasmids were
sequenced before use.
Transfections were with Lipofectamine 2000 (Invitrogen) according to
manufacturer’s protocols. Transient transfections were conducted for 18–24
h. For stable transfections in HeLa and CHO cells, plasmids were subcloned into
the pIRES.hyg3 vector. Cells were kept in a 37 °C incubator at 5% CO2.
Antibodies. Commercial antibodies were monoclonal ␣-FLAG M2 (Sigma),
monoclonal ␣-Actin C4 (ICN Biomedicals), and rabbit polyclonal ␣-ATF6 and
rabbit polyclonal ␣-Grp78 (Santa Cruz). Anti-Gos28 polyclonal antibody is
described elsewhere (26). Anti-ERGIC-53 antiserum was raised against an
Escherichia coli purified GST fusion of amino acids 49–484 in the rat protein.
␣-Ribophorin I was raised against C-terminal peptides corresponding to hu-
man protein (residues 588–605) and mouse protein (residues 576–605). The
Fig. 6. The ATF6 luminal domain controls its localization. (A) Cleavage assay of transiently transfected ATF6 constructs in HeLa cells, either untreated or treated
with 2 mM DTT for 1 h. Red arrow indicates size of the cleaved N-terminal domain. (B) Schematic representation of chimeric proteins with the human Sec22b
cytoplasmic domain (194 aa; yellow), ATF6 transmembrane domain (light blue), and varying ATF6 luminal domains (teal). Proteins contained an N-terminal FLAG
tag. (C) Cleavage assay in HeLa cells transiently transfected with constructs from B. Treatment was for 1 h with 2 mM DTT. (D) Budding reactions on HeLa cells
transiently transfected with Sec22b-ATF6 chimeras. (Upper) Sec22b-ATF6TM; (Lower) Sec22b-ATF6.
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6. ␣-Sec22b was raised against a peptide corresponding to human residues
91–116. The ␣-Sar1 was raised against full-length, thrombin cleaved GST-
fusion protein purified from E. coli.
Cleavage Assay. CHO-ATF6 cells or transfected HeLa cells were grown in
24-well plates to 90% confluency. Media was changed, and 1 h later cells were
treated with toxins for indicated times. For harvesting, wells were washed 1ϫ
with PBS, followed by addition of heated SDS sample buffer
In Vitro Budding Assay. The in vitro vesicle budding assay was performed as
described (27). For description, see SI Methods.
Prebudding Assay. Prebudding complex pulldowns were performed as de-
scribed previously (25) using permeabilized, salt-washed CHO-ATF6 cells. For
description, see SI Methods.
BiP Dissociation and Immunoprecipitation. Permeabilized cells were incubated
for 30 min at 22 °C in the presence of 1 mM ATP or ATP␥S, or 5 mM DTT. Cells
were washed three times and lysed for 1 h in 50 mM Tris, pH 8.0, 150 mM NaCl,
10% glycerol, 2 mM EDTA, and protease inhibitors. Lysates were pelleted at
14,000 ϫ g for 20 min, and supernatants cleared for 1 h with IgG Sepharose.
ATF6:BiP complexes were isolated by incubation with 1 ␮g ␣-FLAG M2 anti-
body conjugated to protein A Sepharose. Control reactions used 1 ␮g mono-
clonal ␣-GFP (Roche).
Immunoblotting and Protein Quantification. Immunoblotting was carried out
according to standard procedures, using either ECL plus enhanced chemilu-
minescence (Amersham) or an Odyssey infrared imaging system (Li-Cor Bio-
sciences) with Alexa Fluor 680 ␣-rabbit (Invitrogen) or IR-dye 800 ␣-mouse
(Molecular Probes) secondary antibodies.
Quantification of protein signal was performed on scanned membranes using
Odyssey software or ImageJ. Budding efficiency was calculated by comparing the
amount in each vesicle lane with the amount in the starting membrane lane.
Prebuddingefficiencywascalculatedbysubtractingthebackgroundsignalinthe
boundSar1-GDPlanefromtheboundSar1-GTPlane,anddividingbytheamount
present in a comparable aliquot of starting lysate. Lysates were loaded at 1% of
total, and bound fractions loaded at 30% of total.
Immunofluorescence Microscopy and Protein Purification. For descriptions, see
SI Methods.
ACKNOWLEDGMENTS. We thank Ron Prywes and Jesse Hay for plasmids;
J. Christopher Fromme, Jinoh Kim, and Bertrand Kleizen for protein purifica-
tion and antibody production; Ann Fischer and Michelle Yasukawa for tissue
culture; and all members of the R.S. laboratory who assisted with experimen-
tal design, data analysis, and manuscript preparation.
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17780 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0910342106 Schindler and Schekman