Zhou et al. 10.1073/pnas.0606480103. |
Fig. 6. Position of the human IRE1a NLD dimer relative to the ER membrane. The NLD dimer is shown in an orientation that is orthogonal to the one in Fig. 2A. Approximately 78 residues are missing from the C terminus of the NLD, represented here as a dashed line. The dark arrow points to an MHC-like groove as previously proposed for binding unfolded polypeptides (1). The program Ribbons was used to produce this figure (2).
1. Credle JJ, Finer-Moore JS, Papa FR, Stroud RM, Walter P (2005) Proc Natl Acad Sci USA 102: 18773-18784.
2. Carson M (1997) Methods Enzymol 277:493-505.
Fig. 7. CD spectra of the human IRE1a NLD. CD indicates that the WT human IRE1a NLD (red line) displays a similar secondary structure with its mutants Q105E (yellow line), D123P (blue line), and W125A (green line).
Fig. 8. CD spectra of the murine PERK NLD. CD indicates that the WT PERK NLD (black line) displays a secondary structure similar to those of its mutants K194P (green line) and L196P (blue line).
Fig. 9. D123P mutant IRE1 is defective in signaling the UPR in S. cerevisiae. The UPR was analyzed by a UPRE-b-Gal reporter assay in yeast strains harboring human-yeast Ire1p (ERN1) chimeras as previously described (1). The pRS316 low-copy expression plasmid harboring the human IRE1a NLD in place of the S. cerevisiae Ire1p NLD was previously described (1). AWY 14, ERN1 WT; AWY 19, ERN1 deleted; WT, AWY 19/pRS316-ERN1-hIRE1a-NLD, WT human-yeast chimera; D123P, AWY 19/pRS316-ERN1-hIRE1a-NLD(D123P), D123P mutant human-yeast chimera. All strains contain the UPRE-b-gal reporter. Cells were propagated, treated with tunicamyin, and analyzed for b-galactosidase activity as described in Supporting Materials and Methods. Whereas the WT chimera rescues the UPR, mutation of D123P at the dimer interface greatly reduces the UPR.
1. Liu CY, Schroder M, Kaufman RJ (2000) J Biol Chem 275:24881-24885.
Fig. 10. D123P mutant IRE1a is defective in Xbp1 mRNA splicing in vivo. (A) Schematic diagram of the expected dicistronic reporter mRNAs produced from pRLIXFL before and after removal of the 26-nt Xbp1 intron. From plasmid pRL-IXFL, the RLuc is translated by ribosome scanning and mXBP1DN/DC(un) (before UPR activation), and mXBP1DN/DC (s)-Fluc (after UPR activation) is translated by IRES-dependent internal initiation, respectively (1). (B) Analysis of Xbp1 mRNA splicing in transiently transfected Ire1a-/- MEFs. Ire1a-/- and WT MEFs were conucleofected with the pRL-IXFL reporter plasmid and plasmids expressing either WT or D123P mutant full-length human IRE1a tagged with Flag epitopes. At 24 h after nucleofection, the cells were further incubated with fresh medium with or without tunicamycin (10 mg/ml) for 8 h. Renilla luciferase and firefly luciferase activities were measured as described in Supporting Materials and Methods. The relative ratio of firefly luciferase to Renilla luciferase activity in each cell lysate was calculated. The columns and bars represent the means and standard deviations of three independent transfection experiments. (C) Expression levels of hIRE1a (WT or D123P) Flag proteins in nucleofected Ire1a-/- MEFs. Western blot analyses were performed by using anti-Flag antibody or anti-actin antibody.
1. Back SH, Lee K, Vink E, Kaufman RJ (2006) J Biol Chem 281:18691-18706.
Fig. 11. Stereo diagram showing the molecular surface of the NLD dimer. The surface is colored white. The cartoon model shows the two underlying monomers colored in yellow and cyan, respectively. The region where symmetry-related Gln-105 residues form a hydrogen bond is marked with a red circle. The program PyMOL (www.pymol.org) was used to create this figure.
Supporting Materials and Methods
Structure Determination and Refinement.
For multiwavelength anomalousdiffraction (MAD) phasing, the positions for five of the seven expected selenium sites were determined by using CNS (1) and further refined by using SHARP (2). One additional selenium site was identified by using the difference Fourier method. Initial MAD phases were calculated with the program SHARP and were further improved with solvent flattening and histogram matching. The initial electron density map was of moderate quality but contained identifiable secondary structural elements. With the assistance of known selenium positions, a partial model was built into the map with program O (3). Phase combination using the partial model and additional rounds of density modification were able to significantly improve the map. Structure refinement consisted of iterations of conjugated gradient minimization, grouped B-factor refinement, torsion angle dynamics simulated annealing using the maximum likelihood target function with experimental phases as a prior phase distribution (MLHL).
Gel Filtration.
The human IRE1a NLD (residues 24-446) and the murine PERK NLD (residues 32-332) were used for gel filtration, sedimentation equilibrium ultracentrifugation, and CD analyses. For each assay, 500 ml of 1.0 mg/ml purified protein was applied to Superdex 200 (Amersham Pharmacia) in a buffer containing 25 mM Tris•HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA, and 1 mM DTT. Peak fractions were analyzed by SDS/PAGE and visualized by Coomassie blue staining.Analytical Ultracentrifugation.
Protein samples were prepared in 20 mM Tris•HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 1.0 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP hydrochloride). Three protein concentrations at OD280 of 0.7, 0.5, and 0.3 were used for each sample. All sedimentation equilibrium experiments were carried out at 4°C by using a Beckman ProteomeLab XL-1 analytical ultracentrifuge, An-50 Ti rotor (Beckman Instruments, Fullerton, CA). Data were collected at seven rotor speeds (7,000, 10,000, 14,000, 20,000, 25,000, 30,000, and 35,000 rpm) and represent the average of 50 scans using a scan step size of 0.001 cm. Data were analyzed by using the UltraScan II program from B. Demeler (University of Texas Health Science Center, San Antonio, TX). The data with the highest concentration equilibrated at 10,000 rpm are converted to a graph of Ln (absorbance) versus radius squared. The slope of this plot is proportional to the molecular weight of the protein. Data are plotted alongside simulations prepared in UltraScan II using theoretical values for molecular weights of the dimer and monomer species and plotted with an arbitrary y axis for direct comparison.CD Measurements.
CD measurements were performed on a Jasco J-810 CD spectropolarimeter at 25°C using a 1-mm cuvette for far UV spectra (195-250 nm). The protein concentration was 0.4 mg•ml-1 in a buffer containing 20 mM KH2PO4 (pH 7.5), 10 mM KCl, and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP hydrochloride). All solutions were centrifuged and degassed before measurement. Spectra represent the average of four runs and have buffer blanks subtracted. Ellipticity values were calculated in degrees•cm2•mol-1 based on an average molecular mass of 111 Da per amino acid residue.Analysis of UPR in S. cerevisiae.
AWY14 and AWY19 yeast strains harboring the UPRE-b-Gal reporter were previously described (4). The pRS316 low-copy expression plasmid harboring the human IRE1a NLD in place of the S. cerevisiae Ire1p NLD was previously described (4). The D123P mutation was introduced into the human-yeast chimera by overlap-extension PCR. AWY19 yeast strains were transformed with the WT or D123P human/yeast chimeric Ire1p expression vectors and selected for growth in synthetic media minus uracil. Yeast cells were grown to mid-log phase in synthetic media minus uracil and treated with 2 mg/ml tunicamycin (Tm) for the indicated periods of time. Cell lysates were prepared by vortexing with glass beads. b-Gal was measured by using the Promega kit E2000. Protein concentration was determined by using the Bio-Rad DC Protein Assay kit.Expression in Mammalian Cells.
The WT human IRE1a expression vector pED-hIRE1a was previously described (5, 6). Overlap extension PCR was used to introduce Flag and HA epitopes at the C terminus of IRE1a. For transient transfection studies, COS1 cells (100-mm plates) were treated with DNA [4 mg of pED-hIRE1a (WT or D123P)-Flag and 4 mg of pED-hIRE1a (WT or D123P)-HA] and 24 ml of FuGENE 6 (Roche) for 30 h. Cells were then incubated in culture medium in the presence or absence of 10 mg/ml Tm for 4 h.Ire1a
-/- MEFs were cultured and nucleofected with the Nucleofector technology according to the manufacturer's instructions (Amaxa Biosystems). Briefly, the primary MEFs (2 ´ 106 cells) were harvested, washed with PBS, and then resuspended in MEF 1 Nucleofector Kit solution with the plasmid DNAs that were linearized by restriction enzyme NdeI digestion. Cells were nucleofected with a human IRE1aWT or mutant expression vector [pEDDC, pED-hIRE1a (WT), pED-hIRE1a (D123P), and pED-hIRE1a (K599A)] (6 mg each) (5, 6) in the presence of the puromycin resistance expression vector pEDPur (2 mg) (5). After nucleofection, cells were cultured for 2 days before applying selection (1 mg/ml puromycin). The puromycin-resistant colonies appearing after 2 weeks were pooled (>100 clones per pool), propagated in puromycin (1 mg/ml) for 1 week, and immediately used for experiments.Analysis of IRE1a
Expression. Logarithmically growing cells were treated with or without 10 mg/ml Tm for 4 or 5 h, as indicated. Cells were washed with ice-cold PBS, harvested in Nonidet P-40 lysis buffer (1% Nonidet P-40/50 mM Tris•Cl, pH 7.5/150 mM NaCl/0.5 mM Na-Vanadate/100 mM NaF/50 mM b-glycerolphosphate/1 mM phenylmethylsulfonylfluoride), supplemented with protease inhibitors (Complete Mini, Roche). Cell lysates were clarified by centrifugation at 13,000 ´ g for 15 min. For analysis of phosphorylated IRE1a, soluble proteins were immunoprecipitated for 6 h with 10 ml of the rabbit anti-IRE1a-specific antiserum (5) bound to 20 ml of protein A agars (Pierce). Where indicated, soluble proteins were immunoprecipitated with 20 ml of EZview red anti-Flag M2 affinity gel (Sigma-Aldrich, St. Louis, MO) for 5 h at 4°C, and precipitates were washed three times for 5 min with Nonidet P-40 lysis buffer. Bound proteins were eluted and resolved by 6% or 10% SDS/PAGE under reducing conditions and transferred to PVDF membranes (Schleicher & Schuell). Immunoblot analyses were performed with mouse anti-IRE1a antibody (1/1,000 dilution) as previously described (7), a 1/500 dilution of alkaline phosphatase-conjugated anti-Flag M2 monoclonal antibody (Sigma-Aldrich), a 1/500 dilution of anti-HA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or a 1/5,000 dilution of anti-actin monoclonal antibody (ICN Biomedical).Quantitative Real-Time RT-PCR Analysis.
Total RNAs were isolated from Tm (10 mg/ml for the indicated times)-treated cell lines by using TRIzol reagent (Invitrogen), and quantitative real-time reverse transcription and PCR analysis for spliced Xbp1 mRNA was carried out as described previously (7).Dual Luciferase Assay.
WT and Ire1a-/- MEFs were cultured and nucleofected with the Nucleofector technology according to the manufacturer's instructions (Amaxa Biosystems). Briefly, the primary MEF cells (2 ´ 106 cells) were harvested, washed with PBS, and then resuspended in the MEF 1 Nucleofector Kit solution with the indicated plasmid DNAs. pED-hIRE1a-Flag (WT), pED-hIRE1a-Flag (D123P), and pEDDC are described in the text. The reporter plasmid pRL-IXFL contains Renilla luciferase (Rluc), the EMCV internal ribosomal entry site, and the murine XBP1 DN/DC (437-576 nt from the unspliced form of murine Xbp1 cDNA sequence) followed by the coding region of firefly luciferase (Fluc) without the first ATG initiation codon (8). After nucelofection, the cell-DNA mixture was divided and cultured into two 10-cm dishes for 24 h. The cells were then further incubated with fresh medium with or without Tm (10 mg/ml) for 8 h. At 32 h after transfection, the cells were washed once with cold PBS, harvested, and stored at -80°C for dual luciferase assay (Promega). Immunoblot analysis using anti-Flag M2 and anti-actin monoclonal antibody were performed as described above.1. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, et al. (1998) Acta Crystallogr D 54:905-921.
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4. Liu CY, Schroder M, Kaufman RJ (2000) J Biol Chem 275:24881-24885.
5. Tirasophon W, Welihinda AA, Kaufman RJ (1998) Genes Dev 12:1812-1824.
6. Tirasophon W, Lee K, Callaghan B, Welihinda A, Kaufman RJ (2000) Genes Dev 14:2725-2736.
7. Back SH, Schroder M, Lee K, Zhang K, Kaufman RJ (2005) Methods 35:395-416.
8. Back SH, Lee K, Vink E, Kaufman RJ (2006) J Biol Chem 281:18691-18706.