Structure and Intermolecular Interactions of the Luminal Dimerization Domain of Human IRE1α
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m300418200
ISSN1083-351X
AutoresChuan Yin Liu, Zhaohui Xu, Randal J. Kaufman,
Tópico(s)Heat shock proteins research
ResumoAccumulation of unfolded proteins in the lumen of the endoplasmic reticulum activates a signal transduction cascade that culminates in the transcriptional induction of genes encoding adaptive functions. One proximal sensor for this unfolded protein response is the protein kinase/endoribonuclease IRE1α. IRE1α is a type-I transmembrane glycoprotein for which the N-terminal luminal domain (NLD) senses the accumulation of unfolded proteins. Previously we demonstrated that the NLD forms a stable ligand-independent dimer linked by disulfide bridges. In this report we have identified the cysteine residues responsible for intermolecular disulfide bonding. However, this covalent interaction was not required for dimerization and/or signaling, suggesting that a cryptic dimer interface exists in the NLD that is independent of covalent disulfide interactions. Limited proteolysis of the NLD revealed characteristic fragments, all retaining the same N-terminal sequences as full-length NLD. Biochemical and functional studies using NLD truncation mutants indicated that the dimerization domain of the NLD is confined to the conserved motifs at the N-terminal regions where putative hydrophobic interactions exist. In addition, the peptide binding domain of the endoplasmic reticulum protein chaperone BiP interacted with the N-terminal region within the NLD. Our findings suggest that the NLD has at least two distinct types of interactions mediating dimerization and function in signaling,i.e. covalent interactions involving disulfide bond formation and hydrophobic interactions, with the hydrophobic interaction being the driving force for dimerization. Accumulation of unfolded proteins in the lumen of the endoplasmic reticulum activates a signal transduction cascade that culminates in the transcriptional induction of genes encoding adaptive functions. One proximal sensor for this unfolded protein response is the protein kinase/endoribonuclease IRE1α. IRE1α is a type-I transmembrane glycoprotein for which the N-terminal luminal domain (NLD) senses the accumulation of unfolded proteins. Previously we demonstrated that the NLD forms a stable ligand-independent dimer linked by disulfide bridges. In this report we have identified the cysteine residues responsible for intermolecular disulfide bonding. However, this covalent interaction was not required for dimerization and/or signaling, suggesting that a cryptic dimer interface exists in the NLD that is independent of covalent disulfide interactions. Limited proteolysis of the NLD revealed characteristic fragments, all retaining the same N-terminal sequences as full-length NLD. Biochemical and functional studies using NLD truncation mutants indicated that the dimerization domain of the NLD is confined to the conserved motifs at the N-terminal regions where putative hydrophobic interactions exist. In addition, the peptide binding domain of the endoplasmic reticulum protein chaperone BiP interacted with the N-terminal region within the NLD. Our findings suggest that the NLD has at least two distinct types of interactions mediating dimerization and function in signaling,i.e. covalent interactions involving disulfide bond formation and hydrophobic interactions, with the hydrophobic interaction being the driving force for dimerization. endoplasmic reticulum N-terminal luminal domain an S-tagged NLD protein a His6-tagged NLD protein unfolded protein response immunoglobulin heavy chain-binding protein peptide-binding domain of BiP nitrilotriacetic acid Caenorhabditis elegans Homo sapiens 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 inSaccharomyces cerevisiae is activated, it functions as a site-specific endoribonuclease (RNase) that splices HAC1mRNA 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, includingKAR2, encoding the polypeptide-binding protein chaperone BiP/GRP78 that is a classical hallmark of UPR activation (1Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (676) Google Scholar). 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 (1Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (676) Google Scholar, 2Mori K. Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 3Kaufman R.J. Scheuner D. Schroder M. Shen X. Lee K. Liu C.Y. Arnold S.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 411-421Crossref PubMed Scopus (502) Google Scholar). 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 (4Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2327) Google Scholar, 5Harding H.P. Zeng H. Zhang Y. Jungries R. Chung P. Plesken H. Sabatini D.D. Ron D. Mol. Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 6Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 7Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (833) Google Scholar). 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 (8Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 9Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). 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 (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To provide an experimental system amenable to study the biochemical and structural basis for transmembrane signaling mediated by the NLD, the entire IRE1α luminal region was produced in a soluble form by transient DNA transfection in COS-1 cells, termed the NLD (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The soluble NLD formed homodimers in a ligand-independent manner. In addition, the NLD interacted with the membrane-bound full-length IRE1α 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. Proteases Glu-C, Lys-C, and trypsin,N-α-tosyl-l-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, and other protease inhibitors were purchased from Roche Applied Science. Mouse α-His5 antibody and Ni-NTA agarose were from Qiagen. Mouse α-NLD antibody was previously described (12Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (746) Google Scholar). S-protein-agarose and S-protein horseradish peroxide conjugate were purchased from Novagen. Dithiothreitol was fromCalbiochem, and β-mercaptoethanol was from Sigma. All other reagents were from Sigma, Fisher, or Calbiochem. The mammalian expression vector of pED-NLD-His6 and pED-NLD-S were described previously (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). DNA fragments corresponding to different C-terminal truncations were amplified and cloned into the XbaI andEcoRI 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 Δ2M (Ser112–Leu441), Δ4M (Leu-185–Leu441), ΔD1 (Ala246–Leu441), or ΔD3 (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 (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). 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-hsIRE1α-NLD, the entire yeast scIre1p-NLD was replaced with the entire NLD of hsIRE1α that was functional for UPR induction (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The DNA fragments corresponding to the truncations of hsIRE1α-NLD including 2M, 3M, 4M, D1, D2, and D4 were amplified and inserted into pRS316-AD at AccIII andPacI 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-hsIRE1α-NLD. for human IRE1α-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 IRE1α-NLD were made: D39N, W54A, D79N, P97A, R158E, and N176Q. BiP Expression Constructs—The wild-type hamster BiP expression vector pEmcBiP was described previously (13Dorner A.J. Wasley L.C. Kaufman R.J. EMBO J. 1992; 11: 1563-1571Crossref PubMed Scopus (299) Google Scholar). 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 (14Morris J.A. Dorner A.J. Edwards C.A. Hendershot L.M. Kaufman R.J. J. Biol. Chem. 1997; 272: 4327-4334Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) 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 anSalI site. Val33 is the 15th residue in the mature BiP protein. The 5′ sense primer used in the PCR contains aSalI site and encodes 410DGDLVLLD, the first 8 amino acids of the PBD. All the 3′ antisense primers contain anXbaI 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 andXbaI sites of pED-BiP-S (I33V). PBD-32K-S encodes the full-length PBD encompassing Asp410–Ser649(β1-β8, αA-αE). 20K-S encodes Asp410–Asp578 (β1-β8, αA), and 15K-S encodes Asp410–Thr527 (β1-β8). 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 mmdithiothreitol. To ensure the specificity of Glu-C, 50 mmammonium 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. 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 mmN-α-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. 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 (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Yeast cell lysates were prepared, and β-galactosidase activity was determined as described previously (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Prediction of potential BiP-binding sites was performed based on previously published methods (15Rudiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Crossref PubMed Scopus (660) Google Scholar, 16Blond-Elguindi S. Cwirla S.E. Dower W.J. Lipshutz R.J. Sprang S.R. Sambrook J.F. Gething M.H. Cell. 1993; 75: 717-728Abstract Full Text PDF PubMed Scopus (567) Google Scholar). 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 (15Rudiger S. Germeroth L. Schneider-Mergener J. Bukau B. EMBO J. 1997; 16: 1501-1507Crossref PubMed Scopus (660) Google Scholar). Previous studies demonstrated that NLD dimer is linked by disulfide bonds (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 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 IRE1α, 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 mmN-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 β-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. 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. 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 andmiddle 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 andmiddle 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, andmiddle, 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. Previously we developed an assay in S. cerevisiae to examine the function of the NLD (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). In this assay system, Δire1 yeast cells harbor a single copy of thelacZ gene under the control of the UPR element fromKAR2. Activation of this β-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 IRE1α while retaining the signal peptide sequence, transmembrane sequence, and the cytoplasmic domain of the scIre1p, the resultant chimeric Ire1p receptor restored UPR-dependent β-galactosidase induction in Δire1. Introduction of the luminal domain of PERK also restored UPR signaling (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Therefore, mutations in the NLD region can be introduced into chimeric Ire1p receptors, and their function can be tested by monitoring UPR-dependent β-galactosidase expression. Receptors carrying single cysteine point mutations in hsIRE1α-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 Δire1 (Fig.2B). Mutation of Cys to Ala in human IRE1α-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. 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) (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 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. 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. 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. 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. 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 (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). 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) (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). 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 hsIRE1α-NLD were generated, and their function in UPR activation was tested using the β-galactosidase reporter assay. Like full-length hsIRE1α-NLD, truncations of D4, D2, D1, 4M, and 3M were all able to inducelacZ 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). 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
Referência(s)