26 Jun Published March 28, 2011 This article has original data
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Published March 28, 2011
This article has original data in the JCB Data Viewer
JCB: Article
http://jcb-dataviewer.rupress.org/jcb/browse/4056
Homeostatic adaptation to endoplasmic reticulum
stress depends on Ire1 kinase activity
Claudia Rubio,1,2 David Pincus,1,2 Alexei Korennykh,1,2 Sebastian Schuck,1,2 Hana El-Samad,2 and Peter Walter1,2
1
Howard Hughes Medical Institute and 2Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158
Here, we uncouple Ire1’s RNase from its kinase activity
and find that cells expressing kinase-inactive Ire1 can
regulate Ire1’s RNase, splice HAC1 mRNA, produce Hac1
protein, and induce UPR target genes. Unlike wild-type
IRE1, kinase-inactive Ire1 cells display defects in Ire1 deactivation. Failure to properly inactivate Ire1 causes chronic
ER stress and reduces cell survival under UPR-inducing
conditions. Thus, Ire1-catalyzed phosphoryl-transfer aids
disassembly of Ire1 signaling complexes and is a critical
component of the UPR homeostatic feedback loop.
Introduction
In eukaryotic cells, all proteins that enter the secretory pathway
must pass through the ER to be properly folded and modified.
When the demand for protein folding in the ER exceeds the
capacity of the compartment, misfolded proteins accumulate
and activate the unfolded protein response (UPR). Activation of
the UPR induces a broad transcriptional program, resulting in
increased production of ER-resident protein folding machinery
and ER-associated degradation components (Travers et al., 2000),
and leading to ER expansion (Bernales et al., 2006; Schuck
et al., 2009). As a consequence, the protein folding capacity of
the ER is increased and protein folding stress is relieved. The
UPR thus serves as a homeostatic feedback loop that monitors
the state of the ER and alters gene expression to adjust protein
folding capacity according to need, thereby restoring proper
function to the ER.
In the yeast Saccharomyces cerevisiae, the UPR is initiated by an ER-resident transmembrane sensor, Ire1 (Cox et al.,
1993; Mori et al., 1993). When activated by the accumulation of
misfolded proteins, Ire1 removes a 252-nucleotide inhibitory
intron from the mRNA encoding Hac1, a bZIP transcription
C. Rubio and D. Pincus contributed equally to this paper.
Correspondence to Peter Walter: [email protected].ucsf.edu
Abbreviations used in this paper: HPL, hyper-phosphorylated loop; SR, splicing reporter; UPR, unfolded protein response; UTR, untranslated region; WT,
wild type.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 193 No. 1 171–184
www.jcb.org/cgi/doi/10.1083/jcb.201007077
factor that up-regulates transcription of UPR target genes
(Cox and Walter 1996; Mori et al., 1996; Travers et al., 2000).
Removal of this intron and ligation of the severed exons by
tRNA ligase produces a spliced form of HAC1 mRNA that is
efficiently translated into the Hac1 transcription factor (Cox and
Walter 1996; Rüegsegger et al., 2001). Because unspliced HAC1
mRNA is not translated before the excision of this intron, Ire1
RNase activation provides the key switch in UPR signaling.
Ire1 is a single-pass transmembrane protein with one domain in the ER lumen and two domains, a kinase and an RNase,
in the cytosol (Cox et al., 1993; Sidrauski and Walter 1997). The
lumenal domain of Ire1 senses unfolded proteins and, once activated, drives Ire1 oligomerization (Shamu and Walter 1996;
Credle et al., 2005). Ire1’s lumenal domain resembles the
peptide-binding domain of antigen-presenting major histocompatibility complexes. We have proposed that direct binding of unfolded polypeptide chains to a presumed peptide binding
groove in this domain provides the activating signal (Credle et al.,
2005; Pincus et al., 2010), although more indirect models of Ire1
activation have also been proposed (Bertolotti et al., 2000;
Okamura et al., 2000; Kimata et al., 2004). Lateral oligomerization
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THE JOURNAL OF CELL BIOLOGY
A
ccumulation of misfolded proteins in the lumen of
the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). Ire1, an ER-resident
transmembrane kinase/RNase, senses the protein folding
status inside the ER. When activated, Ire1 oligomerizes
and trans-autophosphorylates, activating its RNase and
initiating a nonconventional mRNA splicing reaction.
Splicing results in production of the transcription factor
Hac1 that induces UPR target genes; expression of these
genes restores ER homeostasis by increasing its protein
folding capacity and allows abatement of UPR signaling.
© 2011 Rubio et al. This article is distributed under the terms of an Attribution–
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as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
Supplemental Material can be found at:
http://jcb.rupress.org/content/suppl/2011/03/25/jcb.201007077.DC1.html
Original image data can be found at:
http://jcb-dataviewer.rupress.org/jcb/browse/4056
JCB
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Published March 28, 2011
Results
Mutations in Ire1 kinase abolish
phosphoryl-transfer but preserve
RNase activity
Based on sequence conservation between Ire1 and related
CDK2-like kinases as well as the recently solved crystal structures of the cytosolic portion of Ire1 (Lee et al., 2008; Korennykh
et al., 2009), we designed an Ire1 variant with uncoupled kinase and RNase activities. To this end, we identified two catalytic
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residues, D797 and K799, in the nucleotide-binding pocket of
Ire1 kinase. These residues are predicted to coordinate the terminal phosphate of ATP bound to Ire1 kinase (Fig. 1 A), and, by
analogy to other kinases, are required to catalyze phosphotransfer (Lee et al., 2008). We reasoned that mutating these residues
to asparagines would preserve overall steric packing, hydrophobicity, and hydrogen bonding at the kinase-active site but disable
proton transfer and thereby abolish phosphorylation (Fig. S1 A).
Thus, we expected that the mutant Ire1(D797N,K799N) would
be kinase inactive but still able to activate its RNase via nucleotide binding.
To carry out in vitro studies, we recombinantly expressed
and purified the cytosolic portion of Ire1 WT and mutant Ire1.
These constructs consisted of kinase and RNase domains preceded at the N terminus by 32 amino acids derived from the
linker region that tethers the kinase domain to the transmembrane region. We previously showed that this peptide extension
is important, as it enhances Ire1’s ability to activate its RNase
by up to four orders of magnitude (Korennykh et al., 2009).
We term these constructs Ire1KR32 (WT) (Korennykh et al.,
2009) and Ire1KR32(D797N,K799N).
MALDI mass spectrometry analyses have shown that
WT Ire1KR32 is highly phosphorylated when purified from
Escherichia coli, likely as a result of autophosphorylation
(Korennykh et al., 2009). Phosphorylation is evident in the massto-charge ratio (M/z) of WT Ire1KR32, which is higher than
expected based on its theoretical molecular weight (Fig. S1 B).
The shift of 1.3 kD is consistent with the presence of 17
phosphates and can be ameliorated by phosphatase treatment
(Fig. S1 C). In contrast, purified Ire1KR32(D797N,K799N) has
an M/z value that is precisely as expected based on its primary sequence, indicating that this protein is entirely unphosphorylated
(Fig. S1 B; and see Fig. S7 in Korennykh et al., 2009). These
data suggest that Ire1KR32(D797N,K799N) is kinase inactive.
To confirm that Ire1KR32(D797N,K799N) was indeed
kinase inactive, we measured trans-autophosphorylation of the
recombinant proteins in an in vitro kinase assay. As expected,
WT Ire1KR32 showed robust trans-autophosphorylation (Fig. 1 B,
lanes 1–3) whereas Ire1KR32(D797N,K799N) exhibited no detectable kinase activity (Fig. 1 B, lanes 4–6). To show that the
kinase-inactive Ire1 mutant is properly folded and is a competent substrate for phosphorylation, we mixed recombinant
kinase-inactive Ire1 protein with a shorter WT version, Ire1KR,
lacking the 32-amino acid peptide extension (Korennykh et al.,
2009). This enzyme retains WT kinase activity (Fig. 1 B, lanes
7–9) and can be distinguished from the Ire1KR32 versions by
its lower molecular weight. When we mixed Ire1KR in vitro
with Ire1KR32(D797N,K799N), we detected robust phosphorylation of the mutant enzyme (Fig. 1 B, lanes 10–12; top bands).
In these mixing reactions, the top bands corresponding to the
kinase-inactive variant of Ire1 were more extensively labeled
with radioactive phosphate than WT enzyme. This is likely due
to the greater number of unphosphorylated residues in kinaseinactive Ire1 available for phosphorylation when introduced to
kinase-active enzyme.
Based on the previous observation that occupation of
the active site of Ire1 kinase by nucleotide cofactor is sufficient
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brings the cytosolic portion of neighboring Ire1 molecules into
proximity, which promotes trans-autophosphorylation of Ire1
kinase and activation of the RNase (Shamu and Walter 1996).
Mutation of essential catalytic residues and phosphorylation sites in the Ire1 kinase domain block HAC1 mRNA splicing
and prevent up-regulation of UPR target genes (Cox et al., 1993;
Mori et al., 1993; Shamu and Walter 1996), suggesting that
phosphorylation by Ire1 kinase during activation is essential for
RNase function. However, if the nucleotide-binding pocket of
Ire1 kinase is mutated to specifically accommodate the ATPcompetitive drug 1NM-PP1, Ire1 retains RNase activity in response
to ER stress, showing that the requirement for phosphorylation
can be entirely bypassed (Papa et al., 2003). Occupation of the
engineered 1NM-PP1 binding pocket is sufficient to cause the
conformational change in Ire1 that activates the RNase. Because phosphorylation sites are necessary for RNase function
but phosphorylation by itself appears dispensable, the functional significance of phosphoryl-transfer by Ire1 kinase has remained unclear.
Evidence from studies of Ire1-like enzymes supports the
idea that phosphoryl-transfer mediated by the kinase is indeed
dispensable for nuclease activation. RNase L, a close homologue of Ire1, is a cytosolic, ligand-activatable RNase that has
lost kinase activity but retained a catalytically inactive pseudokinase domain (Dong et al., 2001). In contrast, the kinase activity of Ire1 has been preserved in evolution, suggesting a
functional role for Ire1-mediated phosphoryl-transfer. Although
previous findings with 1NM-PP1–sensitized Ire1 kinase are in
apparent contradiction with this idea, those data show only that
Ire1 kinase activity can be bypassed without consequence for
RNase activation; they do not rule out a possible role for the
kinase in the broader scope of UPR biology.
In this study, we explored the role of the Ire1 kinase function in vitro and in vivo by rationally designed, conservative
mutagenesis of central catalytic residues in the Ire1 kinaseactive site. Mutations were designed to preserve interactions
between ATP cofactor and Ire1 but to selectively disrupt catalytic
phosphoryl-transfer. We show that these mutations yield a
kinase-inactive Ire1 that retains wild-type (WT) RNase activity
in living cells. This variant of Ire1 is activated by unfolded protein accumulation without a requirement for exogenous drugs,
such as 1NM-PP1, thereby eliminating potential complications
of off-target effects of the drug within the cell. These studies
confirmed the view that Ire1’s kinase domain regulates its RNase
activity, but also revealed a critical role for phosphoryl-transfer
in the homeostatic feedback of the UPR.
Published March 28, 2011
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Figure 1. Mutations in Ire1 kinase abolish phosphate transfer but preserve RNase activity. (A) A schematic representation of Ire1 depicting the location of
each functional domain. Residues D797 and K799 in the nucleotide-binding pocket of the kinase domain hydrogen bond with the terminal phosphate of
ATP to catalyze phosphate transfer to the substrate serine. Mutation of D797 and K799 to noncatalytic asparagines is predicted to block phosphate transfer
but allow for ATP binding. (B) The kinase activity of recombinant Ire1KR32 (WT, 474 amino acids; lanes 1–3) and Ire1KR32(D797N,K799N) (lanes 4–6)
were measured in an in vitro kinase assay. Recombinant Ire1 was mixed with 0.033 µM [ 32]P-ATP and incubated at 30°C for the time indicated. Reactions
were stopped in 1% SDS loading buffer and separated by SDS-PAGE. A truncated version of WT Ire1, Ire1KR (442 amino acids; lanes 7–9), was mixed
with Ire1KR32(D797N,K799N) (lanes 10–12). (C and D) In vitro RNA cleavage assays were performed using purified substrate RNA, HP21 (C) or Xbp1
(D), and either WT Ire1KR32 or Ire1KR32(D797N,K799N). Reactions were performed in the presence and absence of 2 mM ADP. The lower sensitivity of
the Xbp1 mRNA cleavage reaction to the ADP cofactor during cleavage suggests that a longer RNA substrate may independently stabilize the Ire1 oligomer, perhaps by bridging between multiple adjacent monomers. Bar values were obtained from single-exponential fitting of time courses. Error bars show
standard errors of the single-exponential fitting. The time courses were repeated multiple times and kobs values reproduced within twofold.
Homeostatic adaptation to ER stress • Rubio et al.
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Published March 28, 2011
to cause activation of the RNase, we expected that
Ire1KR32(D797N,K799N) would retain RNase activity and
that its activity would be stimulated by the presence of nucleotide. To test this prediction, we measured RNase activity in an
in vitro cleavage assay using HP21, a previously characterized
small substrate RNA containing a specific Ire1 cleavage site, in
the presence or absence of ADP cofactor. In previous experiments, ADP stimulated Ire1KR32’s RNase activity by 200fold (Korennykh et al., 2009). Here, in the absence of cofactor,
both enzymes exhibited the same basal RNase activity as
Ire1KR32 (Fig. 1 C, “APO”), consistent with previous observations (Korennykh et al., 2009). Addition of ADP increased the
RNase of Ire1KR32(D797N,K799N) 10-fold (versus 100-fold
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JCB • VOLUME 193 • NUMBER 1 • 2011
for WT Ire1KR32; Fig. 1 C, “ADP”). These data are consistent
with the idea that binding of cofactor stimulates the RNase activity of Ire1 in the absence of phosphorylation (Papa et al.,
2003). In in vitro assays using the HP21 substrate, the RNase
activity of Ire1KR32(D797N,K799N) was 10-fold lower than
that of WT. However, when a larger 443-nt Xbp1 mRNA-derived
RNA fragment was used as a substrate (Korennykh et al., 2009),
Ire1KR32(D797N,K799N) cleaved with a rate (kobs = 0.19 s 1)
indistinguishable from that of WT Ire1KR32 (kobs = 0.19 s 1;
Fig. 1 D). The Xbp1 mRNA is a 400-nt substrate derived from
the mammalian counterpart to HAC1 mRNA. This substrate is
cleaved by Ire1 in vitro with kinetics identical to that of HAC1
mRNA substrates of comparable length (unpublished data).
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Figure 2. Ire1 kinase activity, uncoupled from HAC1 mRNA splicing, is important for cell survival during the UPR. (A) Cells bearing WT IRE1 (lanes 1 and 2),
a deletion of ire1 (lanes 3 and 4), or ire1(D797N,K799N) (lanes 5 and 6) were left uninduced ( ) or induced with 2 mM DTT (+). HAC1 mRNA splicing
was analyzed by Northern blotting. The positions of the unspliced (u; 1449 nucleotides) and spliced (s; 1197 nucleotides) forms of HAC1 mRNA are indicated with arrows. Splicing intermediate i1 (980 nucleotides) corresponds to the 5 exon–intron hybrid species, whereas i2 (728 nucleotides) corresponds
to the 5 exon alone. (B) Cells carrying WT IRE1, a deletion of ire1, or ire1(D797N,K799N) were grown in culture, diluted to equal cell number, serially
diluted 1:5, and plated onto permissive medium ( Tm) or medium containing 0.25 µg/ml tunicamycin (+Tm). (C) WT IRE1 or ire1(D797N,K799N) cells
were grown in culture to OD600 0.2, the UPR was induced by the addition of 2 mM DTT. The value for percent viable cells was determined by measuring
the number of colony-forming units over time (see Materials and methods). DTT was refreshed and cells were kept at an OD at or below 0.2 throughout the
duration of the experiment. (D) WT or mutant ire1 cells carrying HA-tagged Hac1 were left uninduced or induced with 2 mM DTT, and total protein was
isolated. Samples were separated by SDS-PAGE and subjected to Western blotting using an anti-HA antibody or an anti-PGK1 antibody. (E) Hac1 protein
was quantified, normalized to PGK1 levels, and plotted. (F) Total protein was isolated from WT- or Ire1(D797N,K799N)- expressing cells, separated by
SDS-PAGE, and subjected to Western blotting using an anti-FLAG antibody. Ire1 protein levels are equivalent in WT and ire1(D797N,K799N) cells.
Published March 28, 2011
The low ADP sensitivity of the Xbp1 mRNA cleavage reaction
suggests a diminished requirement for cofactor during cleavage
of this substrate. Present work in our laboratory is aimed at
understanding the molecular mechanism of this phenomenon.
This longer substrate RNA more closely resembles the endogenous in vivo substrate of Ire1 RNase, suggesting that kinaseinactive Ire1(D797N,K799N) should retain RNase function in
living cells.
Ire1 kinase activity is dispensable for
HAC1 mRNA splicing but enhances cell
survival under ER stress
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Because our in vitro results showed that we had successfully
uncoupled the kinase and RNase functions of Ire1, we used
kinase-inactive Ire1(D797N,K799N) to directly investigate the
role of Ire1 kinase activity in vivo. This approach afforded the
first opportunity to ask this question without requiring the addition of exogenous drug as past studies necessitated.
Our in vitro studies predict that cells expressing
Ire1(D797N,K799N) should splice HAC1 mRNA upon UPR
induction. To test this, we constructed a strain carrying a chromosomally integrated mutant IRE1 allele as the sole copy of
IRE1 in the cell. We then induced the UPR and measured HAC1
mRNA splicing by Northern blotting. We induced ER stress
with DTT, which causes protein misfolding in the ER by disrupting disulfide bond formation. As predicted, spliced HAC1
mRNA was produced upon DTT treatment in ire1(D797N,K799N)
cells (Fig. 2 A, lanes 5 and 6). In contrast, HAC1 mRNA was not
spliced in ire1 cells (Fig. 2 A, lanes 3 and 4). In these experiments, ire1(D797N,K799N) proved mildly hypomorphic, as the
amount of HAC1 mRNA cleaved in the mutant cells was reduced compared with WT and HAC1 splicing intermediates
were more abundant at the time point taken. This was not due to
differences in the expression levels of Ire1 (Fig. 2 F). Nevertheless, these data reinforce the notion that Ire1 kinase activity is
not required for RNA splicing.
We were surprised to discover that splicing of HAC1
mRNA in ire1(D797N,K799N) cells failed to ensure cell survival
under ER stress. When plated on medium containing tunicamycin, a drug that induces the UPR by blocking glycosylation in
the ER, ire1(D797N,K799N) cells displayed a severe growth
defect (Fig. 2 B). This resulted from loss of cell viability rather
than growth arrest: sustained ER stress killed ire1(D797N,K799N)
cells significantly earlier than WT cells (Fig. 2 C).
In search of an explanation for this growth defect, we
tested whether functional Hac1 protein was produced from
spliced HAC1 mRNA in ire1(D797N,K799N) cells. To this end,
we measured Hac1 protein production and determined the scope
of the transcriptional response by assessing global mRNA expression after UPR induction. WT IRE1 or ire1(D797N,K799N)
cells expressing HA-tagged Hac1 were treated with DTT to induce the UPR and probed for HA-Hac1 by Western blotting.
Ire1(D797N,K799N) cells produced Hac1 protein at nearly WT
levels (Fig. 2, D and E). Likewise, the microarray transcriptional profile of UPR-induced ire1(D797N,K799N) cells revealed a profile nearly indistinguishable from that of WT cells
(Fig. S2 A). Canonical UPR target genes were up-regulated
with similar kinetics, and to a comparable extent, in WT and
ire1(D797N,K799N) cells. Specific UPR target genes are highlighted in Fig. 3 A. Collectively, these data show that the observed
reduction in HAC1 mRNA splicing in ire1(D797N,K799N)
cells does not lead to impairment of canonical UPR signaling.
One reason that a cell might die despite expression of
target genes is that mRNAs are not translated. To confirm that
protein products corresponding to UPR targets were also made,
we determined Kar2 protein levels by Western blotting and measured global translation rates during the ER stress. The induction of Kar2 mirrored the microarray result for both WT and
ire1(D797N,K799N) mutant cells (Fig. 3 B), confirming that
expression of this canonical UPR target was intact in both
strains. Furthermore, general translation rates were equivalent
in both WT and ire1(D797N,K799N) cells (Fig. S2 B), indicating that global mRNA translation was not impaired in mutant
cells. No explanation for the enhanced loss of cell viability of
ire1(D797N,K799N) mutant cells was evident in these data.
As a consequence of UPR activation, the ER expands to
meet the increased need for protein folding capacity (Cox et al.,
1997; Bernales et al., 2006; Schuck et al., 2009). To further ensure that UPR signaling downstream of Ire1 was unimpaired,
we measured ER expansion. Using a GFP-tagged version of the
ER marker Sec63 (Prinz et al., 2000), we quantified expansion
of the cortical ER before and after UPR induction in WT and
ire1(D797N,K799N) cells. In confocal sections through the
middle of unstressed cells, the cortical ER marked by Sec63GFP is visible underneath the plasma membrane as a broken
line because the tubular ER network appears in cross section.
Upon ER stress, the cortical ER is converted into expanded
membrane sheets and appears as a continuous line. Consistent
with microarray data showing normal induction of target genes,
UPR-mediated ER expansion occurred normally in mutant cells
(Fig. 3, C and D). Thus, the slight reduction in Hac1 protein
produced in ire1(D797N,K799N) cells (Fig. 2 E) did not weaken
UPR events downstream of Hac1 protein production. Collectively, the data presented thus far indicate that canonical UPR
activation remains intact in ire1(D797N,K799N) cells.
Ire1(D797N,K799N) fails to adapt to
sustained ER stress
The homeostatic feedback response that is mediated by the UPR
is characterized by an activation phase in which Ire1 begins to
signal and an adaptive phase that occurs when cells adjust to ER
stress and Ire1 is turned off (Pincus et al., 2010). Because our
findings indicate that Ire1 activation and induction of its downstream transcriptional targets are normal in ire1(D797N,K799N)
cells, we set out to examine the dynamics of Ire1 activation and
attenuation in ire1(D797N,K799N) cells. To this end, we took
advantage of a splicing reporter, termed SR, previously developed in our laboratory (Aragón et al., 2009). In the SR, the
HAC1 ORF has been replaced by that of GFP (Fig. 4 A), while
the intron as well as the 5 and 3 untranslated regions (UTRs)
of the HAC1 mRNA are maintained so that translational inhibition of SR mimics that of the HAC1 mRNA. Ire1-mediated
splicing of this reporter produces a GFP signal that can be quantitatively measured by flow cytometry.
Homeostatic adaptation to ER stress • Rubio et al.
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Published March 28, 2011
In WT cells, SR fluorescence increased over time with increasing DTT concentration (Fig. 4 B). At low DTT concentrations (below 2 mM), GFP levels in WT cells reached a plateau
after 120 min. This plateau, a result of the long half-life of
GFP, signifies Ire1 deactivation and is characteristic of an intact
homeostatic response that restores the folding capacity of the
ER and quells Ire1 signaling.
In ire1(D797N,K799N) cells, SR splicing in the first
60–120 min was identical to that observed in WT cells. However,
GFP levels continued to rise throughout the time course and
its production continued even at doses of DTT to which WT
cells adapted (Fig. 4 C). This phenomenon was most evident
when reporter activity was plotted as a function of DTT
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JCB • VOLUME 193 • NUMBER 1 • 2011
concentration (Fig. 4, D and E). At the 60-min time point, the
dose–response curves for both WT and ire1(D797N,K799N)
cells overlapped, indicating that GFP production during the
activation phase was equivalent for both WT and mutant enzymes (Fig. 4 D). In marked contrast, at 240 min the curves
deviated substantially (Fig. 4 E), indicating that after prolonged ER stress I…
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