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INTRODUCTION |
The endoplasmic reticulum
(ER)1 monitors the folding
status of newly synthesized secretory and transmembrane proteins and
ensures that only properly folded proteins transit to the Golgi
compartment. In response to accumulation of unfolded proteins in the
ER, cells activate an intracellular signal transduction pathway called
the unfolded protein response (UPR). The yeast UPR is a linear pathway in which the protein kinase/endoribonuclease Ire1p signaling mediates transcriptional activation of UPR target genes. When Ire1p in Saccharomyces cerevisiae is activated, it functions as a
site-specific endoribonuclease (RNase) that splices HAC1
mRNA encoding Hac1p, a basic-leucine zipper-containing
transcription factor. Hac1p binds to the UPR element in the promoter
region and induces transcription of target genes, including
KAR2, encoding the polypeptide-binding protein chaperone
BiP/GRP78 that is a classical hallmark of UPR activation (1).
IRE1, PERK, and ATF6 are the three proximal ER stress transducers that
regulate UPR signaling in metazoan species. IRE1 (yeast scIre1p
homolog) and PERK are two type-I ER transmembrane serine/threonine protein kinase receptors, and ATF6 is a type-II ER
transmembrane-activating transcription factor. The mammalian UPR
includes three adaptive cellular responses that are activated to cope
with the accumulation of unfolded proteins in the ER; 1)
transcriptional induction of ER chaperones and folding catalysts; 2)
transcriptional activation of genes encoding components of
ER-associated protein degradation; and 3) general translational
attenuation (1-3). Recent studies at the organismal level showed that
IRE1 plays critical roles in normal embryogenesis in early development,
and PERK function is required for glucose homeostasis in
vivo (4-7).
IRE1 and PERK are structurally similar to serine/threonine protein
kinase receptors. Dimerization and trans-autophosphorylation is a universal mechanism for activation of this class of cell surface
receptors (8-9). However, the biochemical and structural basis for
this transmembrane signaling in response to conditions of ER stress is
not understood. A biochemical and structural analysis of the NLD should
provide insights into this novel transmembrane signaling event, which
will in turn establish the foundation to understand the physiological
functions of IRE1 and PERK.
IRE1 and PERK contain a remarkably large N-terminal domain that resides
in the ER lumen. The N-terminal luminal domains (NLDs) of IRE1 and PERK
sense the accumulation of unfolded proteins by a common mechanism and
transmit the signal across the ER membrane to induce receptor
activation (10). To provide an experimental system amenable to study
the biochemical and structural basis for transmembrane signaling
mediated by the NLD, the entire IRE1
luminal region was produced in
a soluble form by transient DNA transfection in COS-1 cells, termed the
NLD (11). The soluble NLD formed homodimers in a ligand-independent
manner. In addition, the NLD interacted with the membrane-bound
full-length IRE1
receptor and the ER chaperone BiP. Interestingly,
the NLD homodimer was stabilized by disulfide bridges.
In this report we analyzed the biochemical and structural properties of
the purified NLD homodimer. The cysteine residues responsible for
intermolecular disulfide bond formation were identified, and their
requirement for UPR signaling was examined. The core domain required
for dimerization was defined by limited proteolysis and analysis of
truncation mutants. Our studies demonstrated that sequences required
for dimerization and signaling are confined to conserved motifs at the
N terminus. The existence of a cryptic dimer interface and the
significance of multiple types of intermolecular interactions within
the NLD dimer are discussed.
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EXPERIMENTAL PROCEDURES |
Materials
Proteases Glu-C, Lys-C, and trypsin,
N-
-tosyl-L-lysine chloromethyl ketone,
phenylmethylsulfonyl fluoride, and other protease inhibitors were
purchased from Roche Applied Science. Mouse
-His5 antibody and
Ni-NTA agarose were from Qiagen. Mouse
-NLD antibody was previously
described (12). S-protein-agarose and S-protein horseradish peroxide
conjugate were purchased from Novagen. Dithiothreitol was from
Calbiochem, and
-mercaptoethanol was from Sigma. All other reagents
were from Sigma, Fisher, or Calbiochem.
Construction of Expression Vectors
Mammalian Expression Constructs--
The mammalian expression
vector of pED-NLD-His6 and pED-NLD-S were described
previously (11). DNA fragments corresponding to different C-terminal
truncations were amplified and cloned into the XbaI and
EcoRI sites of the pED vector. The 5' primer used in PCR
reactions contained an XbaI site and encodes MPARRLL of the
N terminus. All the 3' antisense primers contained an EcoRI site at the 3' end and encoded seven specific residues corresponding to
the C terminus of the truncations followed by a His6 tag
and EKDEL sequence for ER retention. The mature NLD protein included 418 amino acid residues (Ser24-Leu441). The
mature C-terminal truncations are D5
(Ser24-Val390), D4
(Ser24-Gly363), D3
(Ser24-Ile334), D2
(Ser24-Val307), D1
(Ser24-Ala246), 4M (4 motifs,
Ser24-Leu185), and 3M (3 motifs,
Ser24-Leu147). All these NLD proteins have a
sequence motif (HHHHHHEKDEL) at the C terminus. To generate N-terminal
truncation mutants, XmaI was introduced to
pED-NLD-C148S/C332S by mutating CCTGAA to
CCCGGG (P29P/E30G). PCR fragments encoding
2M (Ser112-Leu441),
4M
(Leu-185-Leu441),
D1
(Ala246-Leu441), or
D3
(Ile334-Leu-441) were cloned into this vector
at XmaI and EcoRI sites.
Yeast Expression Constructs
pRS316-IRE1,
pRS316-IRE1-AD (AccIII and PacI
sites introduced), C193 and C35 were described previously (10). To make
C6, a pair of primers encoding ISCSNS was designed. The two primers
were annealed and inserted into AccIII and PacI sites in pRS316-AD. In pRS316-AD-hsIRE1
-NLD, the entire yeast scIre1p-NLD was replaced with the entire NLD of hsIRE1
that was functional for UPR induction (10). The DNA fragments corresponding to
the truncations of hsIRE1
-NLD including 2M, 3M, 4M, D1, D2, and D4
were amplified and inserted into pRS316-AD at AccIII and PacI sites to replace the full-length NLD.
Site-directed Mutagenesis
Site-directed mutagenesis by
overlapping PCR was performed to generate single, double, or
triple cysteine mutants in pED-NLD mammalian expression
vector and in pRS316-AD-hsIRE1
-NLD. for human IRE1
-NLD, the
mutants are C109S (TGC
AGC), C148S (TGC
AGC), C332S (TGT
TCT), and N176Q (AAT
CAG). For the NLD of cePERK, the mutants are
C166A (AGT
GCT), C171A (TGT
GCT), and C346A (TGC
GCC). For yeast Ire1p-NLD, the mutants are C263A (TGC
GCC),
C274A (TGT
GCT), and C325A (TGC
GCC). Similarly, point or
double mutants of yeast Ire1p-NLD were made: E129A, W144A, D176A,
P192A, L198A/S202A, D221A, K282A/T283A, and N298Q. Single amino acid
mutants of human IRE1
-NLD were made: D39N, W54A, D79N, P97A, R158E,
and N176Q.
BiP Expression Constructs
The wild-type hamster BiP expression vector
pEmcBiP was described previously (13). A sequence encoding the entire
hamster BiP cDNA was amplified using pEmcBiP as a template. The 5'
sense primer (CTGCAGGACCGCTGAGCACTGGCC) within the 5'-untranslated
region introduced a PstI site. The 3' antisense primer was
designed so that it introduced an XbaI site at the 3' end,
and a 24-amino acid sequence motif was inserted into the C terminus of
BiP between GEEDTS and EKDEL. This 24-amino acid sequence included a
thrombin cleavage site (LVPRGS), an extra glycine residue, and an S-tag
sequence (KETAAAKFERQHMDS). The DNA was cloned into the PstI
and XbaI sites of pED vector to generate pED-BiP-S. To make
pED-BiP-1AD-S, an MscI DNA fragment (850 bp) from
pEmc-1AdelBiP (14) was inserted into the MscI site of
pED-BiP-S to replace the corresponding original sequence. In 1AD
construct, a 27-residue sequence
(Tyr175-Glu201) was deleted from the 1A domain
of ATP binding cleft. To generate expression constructs for the peptide
binding domain (PBD) of BiP, we first constructed pED-BiP-S (I33V), in
which ATCGAC (encoding Ile33-Asp34) was mutated to GTCGAC
(encoding Val33-Asp34) to introduce an
SalI site. Val33 is the 15th residue in the
mature BiP protein. The 5' sense primer used in the PCR contains a
SalI site and encodes 410DGDLVLLD, the first 8 amino acids of the PBD. All the 3' antisense primers contain an
XbaI site at the 3' end and encode 9 amino acids
corresponding to the C terminus of the 32- (32K), 20- (20K), and 15-kDa
(15K) protein, respectively, followed by an S tag and an EKDEL
sequence. These fragments were inserted into SalI and XbaI sites of pED-BiP-S (I33V). PBD-32K-S encodes the
full-length PBD encompassing Asp410-Ser649
(
1-
8,
A-
E). 20K-S encodes
Asp410-Asp578 (
1-
8,
A), and 15K-S
encodes Asp410-Thr527 (
1-
8).
Limited Proteolysis of NLD-N176Q
N176Q was subjected to proteolysis with endoproteinase trypsin,
Lys-C, or Glu-C. Lys-C cleaves at the carboxylic side of lysine. Glu-C,
also known as V8 protease, cleaves specifically at the carboxylic side
of glutamate in the presence of ammonium ion. In the absence of
ammonium ion, it cleaves at both aspartic and glutamic acids. The
reaction buffer for trypsin and Lys-C was 25 mM sodium
phosphate, pH 7.9, 150 mM NaCl, 0.5 mM
dithiothreitol. To ensure the specificity of Glu-C, 50 mM
ammonium bicarbonate was included in the reaction buffer. In a typical
reaction, 20 µl of protein (1 mg/ml) was used in a 50-µl reaction.
The reaction was allowed to proceed at room temperature for 30 min and
then terminated by adding protease inhibitors followed by boiling. Initially the reaction was tested in a series of reactions with protease to protein ratios of 1/2, 1/10, 1/30, 1/100, 1/300, 1/1000. The optimum ratio was 1/30-1/10 for Lys-C, 1/10-1/2 for Glu-C, and
1/300-1/100 for trypsin.
Characterization of Proteolytic Peptide Fragments
The proteolytic fragments were immediately analyzed by SDS-PAGE.
For amino acid sequencing, separated proteins were transferred onto
polyvinylidene difluoride membrane (Schleicher & Schuell) by
electroblotting. The blot was stained with Coomassie Blue R-50 and
destained with 20% methanol. Individual protein bands were excised and
rinsed with water, and dried membrane slices were subjected to
N-terminal amino acid sequencing. For matrix-assisted laser
desorption/ionization mass spectrometry, proteolytic reactions were
stopped by the addition of 1 mM
N-
-tosyl-L-lysine chloromethyl ketone or 1 mM phenylmethylsulfonyl fluoride. Mass spectrometry and
protein sequencing were done either in the Protein and Carbohydrate Structure Facility at the University of Michigan Medical School or in
the Howard Hughes Medical Institute/Keck Biotechnology Resource Laboratory at Yale University.
Other Methods Used in This Study
Transfection of COS-1 cells and cell lysate preparation, protein
binding assay using S-protein-agarose and Ni-NTA agarose, protein gel
electrophoresis, Western blotting, and fast protein liquid
chromatography gel filtration were performed essentially as described
previously (11). Yeast cell lysates were prepared, and
-galactosidase activity was determined as described previously (10).
Prediction of potential BiP-binding sites was performed based on
previously published methods (15, 16). The program for calculating
statistical energy distribution (Gk) to predict
BiP-binding sites was obtained from Dr. Bernd Bukau at the University
of Heidelberg, Berlin, Germany (15).
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RESULTS |
IRE1
Residues Cys148 and Cys332
Participate in Intermolecular Disulfide Bond Formation--
Previous
studies demonstrated that NLD dimer is linked by disulfide bonds (11).
Amino acid sequence alignment showed that there are two highly
conserved cysteines among IRE1 proteins and three among PERK proteins
(Fig. 1A). Interestingly, the
positions of the conserved cysteines between IRE1 and PERK have
diverged. There are three cysteines in human IRE1
, Cys-109, Cys-148,
and Cys-332. To examine the roles of cysteines in the formation of covalently linked dimers, Cys to Ser single mutations were introduced to generate C109S, C148S, and C332S mutants of the NLD. COS-1 cells
were transfected with plasmid DNAs. After 48 h COS-1 cells were
pretreated with 20 mM N-ethylmaleimide, and cell
lysates were prepared in the presence of N-ethylmaleimide.
N-Ethylmaleimide is a membrane-permeable SH-group alkylating
agent. The inclusion of N-ethylmaleimide modifies free SH
groups of cysteines and prevents post-lysis NLD self-association. Cell
extracts were subjected to SDS-PAGE and analyzed by immunoblotting.
Under reducing conditions (100 mM
-mercaptoethanol), all
the NLD mutants were detected as monomers. Under non-reducing
conditions, wild-type NLD and all the mutants were present mostly as
dimers on SDS-PAGE (data not shown). Thus, dimerization appears to
involve more than one intermolecular disulfide bond.

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Fig. 1.
Cys148 and Cys332
residues of human IRE1 participate in
intermolecular disulfide bond formation. A, sequence
alignment showing the conserved cysteine residues in IRE1 and in PERK.
Numerical positions of the cysteine residues are according to hsIRE1
or cePERK. hs, H. sapiens; mm,
Mus musculus; ce, C. elegans.
B, immunoblotting of total cell lysates of double or triple
Cys mutations of NLD-D5. Transfected cell lysates were electrophoresed
either in the presence of 100 mM -mercaptoethanol
(reducing) or under non-reducing conditions on a 7.5% SDS-PAGE gel.
The D5 containing either C148 (lane 4) or C332 (lane
3) or both (lane 2) are linked by intermolecular
disulfide bonds, whereas D5 containing only C109 (lane 5) is
not.
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To identify cysteines that form intermolecular disulfide bonds, mutant
NLD-D5 with two or three mutations from Cys to Ser were made. The
NLD-D5 is a functional truncation of the NLD (see "Discussion"). Cys mutations did not affect protein
expression in transfected COS-1 cells. Under non-reducing conditions,
whereas a majority of wild-type D5 was detected as dimers and higher
multimeric forms by Western blot analysis, a substantial amount of the
double mutants D5-C109S/C148S and C109S/C332S were also present as
dimeric forms (Fig. 1B, lanes 2-4). In contrast,
the double mutant C148S/C332S and triple mutant C109S/C148S/C332S
migrated at a position corresponding to the monomeric form (Fig.
1B, lanes 5-6). These results showed that double
mutations at Cys-148 and Cys-332 completely eliminated the
intermolecular disulfide bonding. The difference in migration between
the different dimeric forms likely represents different conformations
resolved by electrophoresis. Lanes 3 and 4 in
Fig. 1B likely represent homodimers with Cys-332/Cys-332 and
Cys-148/Cys-148 bonds, respectively. Lane 2 in Fig.
1B likely represents a mixture of these two species. Note
that, unlike wild-type D5, the higher multimeric forms were not
observed in the Cys mutants. Taken together, Cys-148 and Cys-332 are
essential for formation of intermolecular disulfide linkages. These
results support that two disulfide bonds bridge each homodimer. One
occurs through Cys-148/Cys-148, and the other occurs through
Cys-332/Cys-332. At this point we cannot rule out that a disulfide
bridge occurs between Cys-148 and Cys-332 in some homodimers.
NLD Dimer Formation Is Independent of Covalent Disulfide
Interactions--
Next we directly examined the ability for these Cys
mutant NLD to form dimers. Wild-type or mutant D5-H
(His6-tagged) were cotransfected with wild-type or mutant
NLD-S (S-tagged). NLD-S bound to S-protein agarose (Fig.
2A, top and
middle panel, pellet, lane 1), whereas wild-type
and mutant D5-H did not (Fig. 2A, bottom panel,
pellet, lanes 1-7). All the D5 constructs were pulled down by S-protein-agarose through their associations with NLD-S or with
NLD-S-C109S/C148S/C332S (Fig. 2A, top and
middle panel). In all the transfection experiments, D5 and
NLD were expressed to a similar level as assessed by Coomassie Blue
staining of total lysate (data not shown). The two asterisks
(Fig. 2A, top, lane 2, and
middle, lane 7) represent NLD and D5 heterodimer
formation in the presence or absence of intermolecular disulfide
bridges, respectively. This result demonstrates that NLD dimer
formation is independent of disulfide bonding. However, elimination of
the two disulfide bridges within the NLD did weaken the subunit
association in the dimer (Fig. 2A, top,
lanes 2-4, middle, lanes 3-7),
suggesting that disulfide interaction within the NLD dimer contributes
to affinity and/or stability. It should be noted that the
intermolecular interactions between NLD and D5-C148S/C332S, between NLD
and D5-C109S/C148S/C332S, and between NLD-C109S/C148S/C332S and D5 were
weak, with only a fraction of D5 proteins pulled down in the assay
(Fig. 2A, top, lanes 5-7,
middle, lane 2). This observation indicates that
dimer formation is favored when the two subunits in the dimeric complex contain either C148/C332 or S148/S332. When one subunit of the homodimer contains C148/C332 and the other contains S148/S332, the
subunit association within the dimer is weaker. The basis and
significance for this observation is not known and awaits structural
determination.

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Fig. 2.
Intermolecular disulfide bonding is not
required for NLD function. A, the
His6-tagged wild-type D5 or D5 mutants (D5-H, 2 µg each) were co-transfected into COS-1 cells with 2 µg of S-tagged
NLD (NLD-S) (top), mutant
NLD-S-(C109S/C148S/C332S) (middle), or pED empty vector
(bottom). Cell lysates were incubated with
S-protein-agarose, and bound protein complexes were analyzed by
SDS-PAGE and silver-staining. Lanes 5 and 6 represent two individually isolated mutant clones of C148S/C332S. The
asterisks in lane 2 and lane 7 indicate two types of heterodimer-forming partners, wild-type NLD·D5
or NLD·D5, with each monomer carrying triple Cys mutations.
B and C, different chimeric Ire1p receptors
carrying wild-type or mutant hsIRE1 -NLD in B and
cePERK-NLD in C were tested for their ability to induce
lacZ expression upon ER stress. K1058A is an RNase mutant of
scIre1p as a negative control. Cells expressing the indicated forms of
receptors were grown to mid-log phase in media lacking uracil and
treated with tunicamycin (2 µg/ml) for 0, 1, or 3 h as
indicated. At each time point, cells were harvested, and extracts were
prepared. Specific -galactosidase activity (milliunits/mg of protein
crude extract) represents an average of three independent assays with
at least two independent clones. Error bars indicate
S.D.
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Intermolecular Disulfide Bonding in the NLD Is Not Required for the
UPR--
Previously we developed an assay in S. cerevisiae to examine the function of the NLD (10). In this assay
system,
ire1 yeast cells harbor a single copy of the
lacZ gene under the control of the UPR element from
KAR2. Activation of this
-galactosidase reporter upon
tunicamycin treatment requires the introduction of a single copy of
wild-type Ire1. When the NLD of yeast Ire1p (scIre1p) was replaced by
the NLD of human IRE1
while retaining the signal peptide sequence,
transmembrane sequence, and the cytoplasmic domain of the scIre1p, the
resultant chimeric Ire1p receptor restored UPR-dependent
-galactosidase induction in
ire1. Introduction of the
luminal domain of PERK also restored UPR signaling (10). Therefore,
mutations in the NLD region can be introduced into chimeric Ire1p
receptors, and their function can be tested by monitoring
UPR-dependent
-galactosidase expression. Receptors carrying single cysteine point mutations in hsIRE1
-NLD,
C109S, C148S, and C332S, were able to sense ER stress in an
identical manner to wild-type NLD (Fig. 2B). The double
cysteine mutant C148S/C332S and the triple mutant C109S/C148S/C332S
also restored Ire1p receptor function in
ire1 (Fig.
2B). Mutation of Cys to Ala in human IRE1
-NLD and in
yeast Ire1p-NLD (C263A, C274A, and C325A) did not reduce receptor
signaling (data not shown). Finally, receptors carrying Cys mutations
in the cePERK-NLD including C166A/C171A, C346A and C166A/C171A/C346A
were also functional in replacing the scIre1p-NLD (Fig.
2C).
These results demonstrated that intermolecular disulfide bonds are not
required for either receptor dimerization or UPR signaling upon ER
stress. Our data suggest that the driving force for NLD dimerization is
from interactions other than intermolecular disulfide bonds.
Proteolytic Cleavage Analysis of the NLD--
To identify the
molecular interactions responsible for dimerization and to understand
the structural organization of the NLD, we performed limited
proteolysis of the non-glycosylated NLD-N176Q. Limited proteolysis by
Lys-C gave rise to one major and two minor bands (Fig.
3A, 3,
6, and 7). Proteinase Glu-C digestion also generated one major and two minor, albeit different bands (Fig. 3A, 1, 2, and 4). Trypsin
digestion resulted in yet another different major band (Fig.
3A, 5) and five other minor bands. Western
blotting confirmed that all these trypsinized bands originated from the NLD (Fig. 3B). Each of the three major bands and two
relatively strong minor bands were excised from a polyvinylidene
difluoride blot and sequenced at the N-terminal end by Edman
degradation. The N-terminal amino acid sequences of all the five
proteolytic fragments were STVTLPETLL, identical to that of the
full-length NLD (Fig. 3C) (11). It should be noted that
there are 32 Glu and 28 Lys residues that are distributed throughout
the entire NLD sequences. These results indicate that the NLD possesses
relatively few sites accessible to proteases despite many potential
cleavage sites.

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Fig. 3.
Proteolytic analysis of the soluble
NLD-N176Q. A and B, proteolytic cleavage of
NLD-N176Q with Lys-C, Glu-C, or trypsin generated characteristic
fragments. The proteolytic reaction was analyzed on a 4-15% gradient
SDS-PAGE and visualized by Coomassie Blue staining. The trypsinized
fragments were confirmed in B by Western blotting with
-NLD antibody. Fragments 1-7 are the major or strong minor cleavage
products. C, proteolytic cleavage mapping of the NLD-N176Q.
The full-length N176Q is presented as a thin line, and
mature proteins are shown as a dot-filled rectangles. The
full-length protein contains 442HHHHHHEKDEL452
at the C terminus as represented by a filled rectangle.
Met1-Thr23 is the signal peptide. The five
fragments (1-5) from A were sequenced, and their molecular
masses were determined. The vertical arrows indicate these
five cleavage sites.
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All five bands were subjected to analysis by mass spectroscopy. The
determined molecular masses were 41.2, 39.9, 39.0, 34.5, and 30.3 kDa.
Based on their masses, we mapped five major accessible cleavage sites
to Glu388, Glu377, Lys374,
Glu331, and Lys288, respectively (Fig.
3C). The fact that all the major protease accessible sites
are located at the C-terminal part of the NLD indicated that the
C-terminal part of the molecule is much more flexible than the
N-terminal part. In addition, mass spectroscopy analysis showed that
the three major proteolytic fragments by Glu-C were dimers (data not
shown). Therefore, the N-terminal region is likely to contain the core
structure for dimerization.
C-terminal Truncation Mutants Can Form Dimers--
To test the
hypothesis that the core structure for NLD dimerization is located at
the N terminus, we constructed C-terminal truncation mutants of the
NLD: D5, D4, D3, D2, D1, 4M, and 3M (Fig.
4A). Truncations were
expressed in COS-1 cells by transient DNA transfection. The D5 was
expressed to a level comparable with full-length NLD, and expression
levels of D3 and D4 were lower. Surprisingly, D2 and D1 expression
levels were significantly lower than D3 (Fig. 4B), and the
expression of 4M and 3M could not be detected (data not shown).
Although the mechanism for this decreased protein expression is not
examined in this study, it is likely that the C-terminal regions may
contribute to the stability of the soluble NLD.

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Fig. 4.
The C-terminal truncations of the NLD can
form dimers. A, schematic diagram of the C-terminal
truncation constructs of the NLD. All the mature NLD proteins contain a
Ser24 at the N terminus and an 11-residue sequence
(HHHHHHEKDEL) at the C terminus as indicated by a filled
rectangle. Their relative expression in transfected cells is shown
on the right. B, analysis of transfected cell lysates by
Coomassie Blue staining showing the relative expression of D1, D2, and
D3. C, the NLDs deleted of the C-terminal regions can form
heterodimers with full-length NLD. The full-length NLD-S (S-tagged) (2 µg) was co-transfected into COS-1 cells with NLD truncation
constructs. Two µg of plasmid DNA was used for D3, D4, and D5, and 4 µg was used for D1 and D2. Cell lysates were incubated with
S-protein-agarose, and bound protein complexes were analyzed by
SDS-PAGE and silver-staining. Note that the endogenous BiP was pulled
down through its association with the NLD-S.
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These deletion mutants were used to test for their ability to form
heterodimers with full-length NLD. NLD-S (S-tagged) was co-transfected
into COS-1 cells with the truncation mutants, and heterodimer formation
was detected by an S-protein binding assay. All the five truncations
were pulled down by S-protein-agarose through their interaction with
NLD-S (Fig. 4C, lanes 7-11). In the absence of
NLD-S, no interacting protein was detected (Fig. 4C,
lanes 2-6). Because 3M and 4M did not express well in
transfected cells, their dimerization with the NLD was not examined.
These results demonstrated that the N-terminal region of the NLD can form dimers.
The Conserved Motifs at the N Terminus of the NLD Are Sufficient
and Required for the UPR--
Within the N-terminal region of the NLD
there are four motifs that are conserved among IRE1 homologues from
different species. These motifs are also conserved in the NLD of PERK
(10). Therefore, we asked whether the N-terminal region of the NLD
alone is able to signal the UPR. First of all, removal of the
N-terminal region of the NLD or the entire luminal domain from Ire1p
receptor abolished its ability to induce the UPR (Fig.
5A) (10). It is noted that none of the N-terminal deletion mutants contains the four conserved motifs. To define the sequence requirement for NLD function, Ire1p receptors containing deletions from the C terminus of hsIRE1
-NLD were generated, and their function in UPR activation was tested using
the
-galactosidase reporter assay. Like full-length hsIRE1
-NLD, truncations of D4, D2, D1, 4M, and 3M were all able to induce lacZ expression upon ER stress. However, 2M was defective in
UPR induction, suggesting that deletion of motif 3 and motif 4 abolished NLD function and Ire1p signaling (Fig. 5B).

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Fig. 5.
The three conserved motifs at the N terminus
of the NLD are necessary and sufficient for UPR signaling.
A, deletion of the conserved N-terminal motifs of the NLD
from yeast scIre1p receptor abolished the UPR. Deletion constructs are
shown on the left. B, the three conserved motifs
at the N terminus of hsIRE1 -NLD are sufficient and required for the
UPR. Deletion constructs are shown on the left. The scIre1p
signal peptide (SP), transmembrane domain (TM),
and cytoplasmic domain (CD) are indicated. The scIre1p-NLD
is shown as an open rectangle in A, and the
hsIRE1 -NLD is shown as a dot-filled rectangle in
B. K1058A carries a mutation in the RNase domain and is an
inactive form of scIre1p. C, the highly conserved residues
within the four motifs of scIre1p-NLD are not critical for NLD
function. All the experiments were carried out essentially the same as
in Fig. 2, B and C.
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There are only about 16 identical/similar residues (~6%) among the
NLDs of IRE1 and PERK including five glycines, and yet both NLDs were
able to substitute for yeast Ire1p-NLD to signal the UPR. All of the
highly conserved residues are localized to the four motifs at the N
terminus of the NLD (10). To test the requirement of the conserved
residues, they were each singly or doubly mutated to alanine in
scIre1p-NLD. All of the single or double mutant receptors constructed
were able to restore tunicamycin-dependent
-galactosidase induction in
ire1 cells (Fig.
5C) (10). Similarly, single mutations of the conserved
residues of human IRE1
-NLD including D39N, W54A, D79N, P97A, R158E,
and N176Q did not affect human NLD function in inducing the UPR when
compared with wild-type human IRE1
-NLD (data not shown).
Taken together, these results demonstrated that the three conserved
motifs at the N terminus are sufficient and required for dimerization
and UPR induction. It is interesting to note, however, that neither of
the conserved residues within this region of the NLD is critical for
NLD structure and/or function.
The Peptide Binding Domain of BiP Interacts with the NLD--
We
asked whether BiP interacts with the NLD. BiP has two major domains,
the N-terminal ATPase domain with no peptide affinity and the
C-terminal PBD. The crystal structures of ATPase domain from bovine
HSC70 and of PBD-peptide complex of Escherichia coli DnaK
aided our understanding of the mechanism of BiP function (17-18). The
ATPase domain has a four-domain structure with the nucleotide bound in
a deep cleft. The PBD consists of a compact
sandwich followed by
helical elements, and the peptide substrate is bound in an extended
conformation in the
sandwich. The
helical domain acts as a
hinged lid to regulate the peptide binding and release in communication
with the ATPase domain. We first constructed wild-type hamster BiP
expression constructs, pED-BiP-S (S-tagged). 1AD-S is a 27-amino acid
deletion mutant of the 1A domain of the ATP binding cleft. Although 1AD
is capable of binding ATP and immunoglobulin heavy chain, this mutant
BiP has no ATPase activity (19). 32K-S is the full-length PDB without
the ATPase domain containing all the eight
-strands (
1-
8) and
five
-helices (
A-
E). The 20K-S contains
1-
8 and
A,
and 15K-S contains only
1-
8.
Wild-type BiP interacted with D5 and D3 by a protein binding assay
using S-protein-agarose (Fig. 6,
A, lane 6, and B, lane 2).
Mutant 1AD also interacts with D3 (Fig. 6B, lane
3), D4, and D5 (data not shown). Second, all the three PBD
constructs, 32K, 20K, and 15K, bound to D5 or D3 (Fig. 6, A,
lanes 7-9, and B, lanes 4-6). Third, although
the interaction between BiP and D2 was detectable (Fig. 6C,
lanes 1-3), the interaction was much weaker compared with
that of D3 (Fig. 6C, lanes 4-6). The amounts of
D2 and D3 proteins bound to Ni-NTA beads represent their expression levels in total cell lysates (Fig. 6C, bottom
panel). The interaction between D1 and BiP was weak also (data not
shown). We were unable to obtain 4M protein in COS-1 cells, and
therefore, its interaction with BiP was not examined. Under our
experimental washing conditions, all the NLD truncations from D1 to D5
did not bind to S-protein-agarose to a detectable level by either
silver-staining or Western blot analysis (Fig. 6, A,
lane 1, B, lane 1, and D
and C, lanes 2-6). Our results showed that the
PBD of BiP interacts with the N-terminal regions of the NLD. However,
the Val307-Ile334 region, which is absent in
D2 but present in D3, contributes to high affinity BiP binding.

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Fig. 6.
The peptide binding domain of BiP interacts
with the N-terminal region of the NLD. A and
B, the PBD of BiP interacts with both D3 and D5.
His6-tagged D5 (D5-H) in A or D3
(D3-H) in B (2 µg each) were co-transfected
into COS-1 cells with S-tagged BiP constructs as indicated (3 µg
each, except 6 µg for 1AD). Cell lysates were incubated with
S-protein-agarose, and bound protein or protein complexes were analyzed
by Western blotting using mouse -His5 antibody and S-protein
horseradish peroxide conjugate ( -S). D5 and D3 expression was
examined by Western blot (IB) analysis as shown in the
lower panel. 1AD expression was significantly lower compared
with other BiP constructs. C, the interaction of PBD with D2
is weaker compared with that of D3. Pull-down percentages (%) indicate
the ratio of D2 or D3 protein that were pulled down by
S-protein-agarose relative to total D2 or D3 protein in cell lysates,
respectively. Protein quantitative analysis was performed using
Quantity One software (Bio-Rad). Protein binding analysis was performed
using S-protein-agarose and Ni-NTA agarose, and bound proteins were
analyzed by silver-staining. D2 and D3 proteins bound on Ni-NTA reflect
their relative expression in transfected cells. D, as a
control, single transfected 32K-S did not bind to Ni-NTA, and D2-H and
D3-H did not bind to S-protein agarose.
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Potential BiP-binding Sites in the NLDs of Human IRE1
and PERK
Are Distributed Similarly--
To further analyze potential
BiP-binding sites within the NLD, we used an algorithm based on peptide
binding motifs for DnaK identified by an extensive peptide scan (15).
This algorithm allows for prediction of DnaK-binding sites with high
accuracy in natural proteins by sequence alignment. The DnaK binding
motif consists of 13 residues, a central hydrophobic core of 5 residues, and two 4-residue flanking regions. The nature of this
consensus DnaK recognition motif is similar to the extended hydrophobic heptapeptide consensus BiP binding motif previously described (16).
This algorithm is applicable to BiP also because the general features
of the peptide-binding sites of HSP70 family proteins are conserved. In
particular, all of the amino acids that contact the bound peptides in
DnaK are conserved in BiP (18). This algorithm calculates the
statistical energy distribution (
Gk) of each
amino acid in the motif. 
Gk is a measure of the potential for a particular peptide to bind BiP. The lower the

Gk value obtained for a specific segment, the
higher the predicted affinity for BiP. A positive score by this scoring system indicates the peptide motif has a low probability to bind to
BiP, whereas scores below
5 indicate a significant probability of
binding to BiP (15). In the NLD of human IRE1
and human PERK, four
motifs (M1-4) at the N terminus and three other regions (R5-7) at the
C terminus were found to contain peptides with scores below or close to
5 (dotted line) and, therefore, represent potential BiP-binding sites (Fig. 7A).
Consistent with this finding, these sites are also predicted to be
hydrophobic in nature (data not shown). However, it remains to be
determined if any of these predicted sites are in a binding-competent
conformation in the folded native NLD. Sequence alignment suggested
that these potential BiP binding regions are similarly distributed
between IRE1 and PERK (Fig. 7B). The four conserved motifs
at the N terminus each contain one or more potential BiP-binding sites,
and the positions of these sites are also well conserved. A number of
potential BiP-binding sites are distributed throughout the
non-conserved C-terminal region of the NLD. These sites can be grouped
into regions 5, 6, and 7, although their positions are not well
conserved (Fig. 7B). In summary, although the primary
sequences of the NLDs of IRE1 and PERK have diverged, they share a
common feature of hydrophobicity and multiple potential BiP-binding
sites, which may provide the biochemical basis for a common mechanism
of dimerization and function.

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Fig. 7.
Potential BiP-binding sites in the NLDs of
human IRE1 and PERK are distributed
similarly. A, multiple potential BiP-binding sites were
identified by averaged energy distribution
( Gk) analysis. Four motifs (M1-4) and three
other regions (R5-7) were found to contain peptides with scores below
5 (dotted line). Energy scores of 5 or lower possess
significant probability of binding to BiP (15). The x axis
represents relative residue numbers where the first residue is
Ser24 for human IRE1 -NLD and Glu96 for human
PERK-NLD. B, sequence alignment of conserved BiP binding
regions between human IRE1 and PERK. The five-residue hydrophobic
core for each potential BiP-binding site is underlined, and
the  Gk values are shown above the
core sequence for IRE1 and below the core sequence for PERK.
Dots denote identical/similar residues. Dashes
represent omitted sequences, and spaces represent gaps to
obtain maximum sequence alignment. Arrows show the three
truncations of IRE1 -NLD (D1, D2, and D3) and a deletion mutant of
PERK-NLD ( 4).
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DISCUSSION |
The NLD Dimerizes through Hydrophobic Interactions--
In this
study we analyzed the molecular nature by which the luminal domain of
IRE1
forms dimers. Both full-length IRE1
receptor and the NLD
form homodimers through intermolecular disulfide bonds (11). However,
mutants carrying Cys
Ser mutations did not disrupt dimer formation
and UPR induction, suggesting that a cryptic dimer interface exists
that is independent of the covalent interaction. To probe the subdomain
structure of the NLD, we performed limited proteolysis. The results
indicated that all the major sites accessible to cleavage are located
at the C-terminal region of the NLD. C-terminal-truncated forms of the
NLD were functional in their ability to associate with full-length NLD
and to induce the UPR in yeast. Furthermore, we showed that deletion of
the conserved motifs from the N terminus abolished UPR signaling. In
search for the interactions responsible for dimerization, we
demonstrated that the cysteine-less NLD dimer is sensitive to SDS
detergent, indicative of noncovalent interactions such as hydrophobic
and electrostatic interactions. We showed that the N-terminal fragment
(D1, Ser24-Ala246) formed dimers and was
functional. Although the low level expression and/or instability
precluded analysis of dimer status of shorter truncations, 3M
(Ser24-Leu147), which constitutes the first
three conserved motifs at the N terminus, was functional in sensing ER
stress to signal the UPR. We conclude that the NLD dimerizes mainly
through these three conserved motifs at the N terminus. Previously we
demonstrated that a basic-leucine zipper dimerization motif conferred
ER-stress inducible Ire1p receptor activation and UPR induction (10). This implies that dimerization and the subsequent receptor activation can occur simply through hydrophobic interactions within the leucine zipper. It is interesting to note that the conserved motifs in the NLDs
of IRE1 and PERK are characterized by an abundance of hydrophobic amino
acids and hydrophobic motifs. Taken together, we propose that
hydrophobic interactions within the N terminus of the NLD, rather than
intermolecular disulfide bonding, are the driving force for dimer formation.
The NLD Evolved as a Robust ER Stress Sensor--
The ER is a
unique protein-folding compartment and differs from cytosol in its high
oxidizing potential, high Ca2+ concentration, and the
presence of glycosylation machinery. Perturbation of this unique
environment elicits the up-regulation of chaperones and folding
catalysts that are present at basal levels under normal conditions. Our
results support that the NLD structure is not sensitive to conditions
that activate the UPR. First, because NLD dimerization does not depend
upon covalent disulfide interactions, its function will not be
disturbed by reductive stresses such as dithiothreitol or
-mercaptoethanol treatment. Second, although IRE1
is a
glycoprotein, NLD glycosylation is not essential for dimer formation or
UPR signaling. This allows the NLD to function even under conditions
that disrupt oligosaccharide addition and/or removal. Third, the
maintenance of the purified dimeric receptor does not require
nucleotides such as Mg2+-ATP or metal ions such as
Ca2+, for which the ER is the major storage site (11).
Thus, the function of the NLD is not sensitive to fluctuations in ATP
or calcium concentrations in the ER. Fourth, the NLD can tolerate amino
acid mutations of conserved residues and even extensive deletions from
the C terminus. The only region within IRE1 and PERK that is conserved,
although very weakly, is at the N terminus of the NLD. A common feature
between these regions is the hydrophobic character of amino acids that
may provide a hydrophobic interface for dimerization. This may account
for the ability for these two divergent domains to respond to the same
ER stress despite their low sequence homology. Finally, the NLD
displays high affinity self-association, also suggesting that a large
hydrophobic dimer interface exists. Indeed, prediction of potential
BiP-binding sites identified extensive hydrophobic regions in the
conserved N-terminal motifs (see below). Our data demonstrate that the
NLD can tolerate a local disturbance of its three-dimensional framework without disrupting the dimer interface. This ensures that the NLD
maintains structural integrity and function even in the presence of
mutations under conditions that reduce the fidelity of protein translation or under conditions that disrupt protein folding in the ER.
We, therefore, conclude that the NLD has evolved as a robust ER stress
sensor, suitable for responding to conditions that induce protein
misfolding in the ER.
Molecular Nature of UPR Regulation by BiP--
BiP is a negative
regulator of the UPR and interacts with all three ER stress sensors,
IRE1, PERK, and ATF6, under non-stress conditions (10-11, 20-23).
Upon accumulation of unfolded proteins in the ER, these stress sensors
are released from BiP to initiate downstream signaling. It is proposed
that unfolded proteins bind and sequester BiP so that it is no longer
available for interaction with IRE1, PERK, and ATF6. Upon release from
BiP, ATF6 transits to the Golgi compartment, where it is processed to
its transcriptionally active form (21). The release of IRE1 and PERK
from BiP promotes their respective homodimerization and
trans-autophosphorylation for activation. In this manner BiP
senses unfolded proteins in the ER lumen and activates the UPR.
Although BiP is the master regulator of UPR activation, the molecular
nature of its interaction with the luminal domains of the stress
transducers remains to be defined. Three distinct regions within the
ATF6 luminal domain were identified that have different affinities for
BiP (23). A region (Lys411-Leu481) in human
PERK-NLD was identified as a strong BiP-binding site (20). In the
current study, we analyzed interaction of IRE1
-NLD truncations with
BiP to conclude that BiP interacts with the N-terminal region of the
NLD, with apparent higher affinity to fragments that contain residues
Val307-Ile334. Interestingly, both the
Val307-Ile334 region of IRE1
-NLD and the
4 region (Lys411-Leu481) of PERK-NLD are in
region 6. This region in human IRE1
-NLD is not required for
signaling the UPR in yeast (10). It remains to be determined whether
this region in human PERK is required to confer ER
stress-dependent UPR activation.
Our studies also demonstrated that the NLD binds to the eight-stranded
compact
sandwich within the peptide binding domain of BiP
(Asp410-Thr527). Therefore, we assume that the
NLD binds to the peptide binding pocket in BiP. Our results support
that there are multiple regulatory BiP-binding sites in the NLD. and
these sites may be functionally redundant. BiP binding to the
N-terminal region of the IRE1-NLD may be functionally significant. Our
studies suggest that the Val307-Ile334 motif
binds BiP with high affinity. Interestingly, proteolytic and deletion
analysis showed that the Val307-Ile334 motif
is not present in the dimer interface, allowing its association with
BiP. It is, therefore, likely that hydrophobic motifs within the N
terminus up to residue Val307 form a dimer interface, and
the Val307-Ile334 motif is an exposed site
available for BiP binding. To test this hypothesis, we constructed
N-terminal deletion mutants of the NLD,
2M
(Ser112-Leu441),
4M
(Leu185-Leu441),
D1
(Ala246-Leu441), and
D3
(Ile334-Leu441). We showed that all these
truncations also interacted with BiP (data not shown), although
sequences in
4M were not required for the UPR. It remains to be
examined whether these regions including the
Val307-Ile334 motif are the bona
fide BiP-binding sites in vivo for regulating NLD
dimerization. We speculate that the weak BiP interaction with the
N-terminal region, compared with the high affinity homodimer association, may be sufficient for BiP to regulate IRE1 dimerization. The great excess of endogenous BiP over the level of endogenous IRE1
provides a kinetic advantage for BiP to interact with the NLD. The high
affinity self-association of the NLD would provide the driving force
for receptor dimerization and activation under conditions of ER stress.
In summary, we have identified cysteine residues responsible for
intermolecular disulfide bond formation. We showed both biochemically and functionally that removal of the intermolecular disulfide linkages
does not affect receptor dimerization or its function in UPR signaling.
The structural organization of the NLD was defined by limited
proteolysis and functional analysis. We showed that the functional
dimerization domain is located at the N terminus of the NLD. The
characterization of the NLD permitted the production of functional
truncations that are neither glycosylated nor covalently disulfide-linked. These proteins should prove useful in generating crystals for structure determination of IRE1 to elucidate how the
accumulation of unfolded proteins in the ER leads to receptor activation.