Rapid Turnover of Unspliced Xbp-1 as a Factor That Modulates the Unfolded Protein Response

The mammalian and yeast unfolded protein responses (UPR) share the characteristic of rapid elimination of unspliced Xbp-1 (Xbp-1u) and unspliced Hac1p, respectively. These polypeptides derive from mRNAs, whose splicing is induced upon onset of the UPR, so as to allow synthesis of transcription factors essential for execution of the UPR itself. Whereas in yeast translation of unspliced Hac1p is blocked, mammalian Xbp-1u is synthesized constitutively and eliminated by rapid proteasomal degradation. Here we show that the rate of Xbp-1u degradation approaches its rate of synthesis. The C terminus of XBP-1u ensures its trafficking to the cytoplasm, and is sufficient to impose rapid degradation. Degradation of XBP-1u involves both ubiquitin-dependent and ubiquitin-independent mechanisms, which might explain its unusually rapid turnover. Xbp-1-/- mouse embryonic fibroblasts reconstituted with mutants of XBP-1u that show improved stability differentially activate UPR target genes. Unexpectedly, we found that one of the mutants activates transcription of both Xbp-1-specific and non-Xbp-1-dependent UPR targets in response to tunicamycin treatment, even more potently than does wild type Xbp-1. We suggest that the degradation of Xbp-1u is required to prevent uncontrolled activation of the UPR while allowing short dwell times for initiation of this response. The mammalian and yeast unfolded protein responses (UPR) share the characteristic of rapid elimination of unspliced Xbp-1 (Xbp-1u) and unspliced Hac1p, respectively. These polypeptides derive from mRNAs, whose splicing is induced upon onset of the UPR, so as to allow synthesis of transcription factors essential for execution of the UPR itself. Whereas in yeast translation of unspliced Hac1p is blocked, mammalian Xbp-1u is synthesized constitutively and eliminated by rapid proteasomal degradation. Here we show that the rate of Xbp-1u degradation approaches its rate of synthesis. The C terminus of XBP-1u ensures its trafficking to the cytoplasm, and is sufficient to impose rapid degradation. Degradation of XBP-1u involves both ubiquitin-dependent and ubiquitin-independent mechanisms, which might explain its unusually rapid turnover. Xbp-1-/- mouse embryonic fibroblasts reconstituted with mutants of XBP-1u that show improved stability differentially activate UPR target genes. Unexpectedly, we found that one of the mutants activates transcription of both Xbp-1-specific and non-Xbp-1-dependent UPR targets in response to tunicamycin treatment, even more potently than does wild type Xbp-1. We suggest that the degradation of Xbp-1u is required to prevent uncontrolled activation of the UPR while allowing short dwell times for initiation of this response. Protein folding in the endoplasmic reticulum (ER) 4The abbreviations used are: ER, endoplasmic reticulum; MEF, mouse embryonal fibro-blasts; UPR, unfolded protein response; Ub, ubiquitin; Xbp-1, X-box binding protein 1; Xbp-1u, unspliced X-box binding protein 1; Xbp-1s, spliced X-box binding protein 1; GFP, green fluorescent protein; HA, hemagglutinin; WT, wild type. 4The abbreviations used are: ER, endoplasmic reticulum; MEF, mouse embryonal fibro-blasts; UPR, unfolded protein response; Ub, ubiquitin; Xbp-1, X-box binding protein 1; Xbp-1u, unspliced X-box binding protein 1; Xbp-1s, spliced X-box binding protein 1; GFP, green fluorescent protein; HA, hemagglutinin; WT, wild type. is carried out under the constant scrutiny of the ER quality control machinery (1Ellgaard L. Helenius A. Curr. Opin. Cell Biol. 2001; 13: 431-437Crossref PubMed Scopus (332) Google Scholar). The overall capacity of the ER to fold newly synthesized proteins must match the load of client proteins that emerge into the ER. When this amount exceeds the folding capacity of the ER, a signaling pathway emanates from the ER that controls gene transcription, as well as protein translation. This ER to nucleus signaling cascade is referred to as the unfolded protein response (UPR). The overall goal of the UPR is to enhance the clearance of misfolded proteins from the ER, and consequently the UPR alleviates ER stress (2Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar). In yeast, Ire1p is the only known transducer of the UPR. In response to ER stress conditions, Ire1p dimerizes and undergoes autophosphorylation. This event induces a conformational change that activates a nuclease domain located in its cytosolic tail (3Sidrauski C. Walter P. Cell. 1997; 90: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 4Papa F.R. Zhang C. Shokat K. Walter P. Science. 2003; 302: 1533-1537Crossref PubMed Scopus (195) Google Scholar). By means of this nuclease activity, activated Ire1p splices the mRNA of Hac1, which in its spliced form encodes Hac1p, a potent transcription factor that induces transcription of many genes that encode ER chaperones, proteins that participate in ER to Golgi trafficking and components of the ER degradation machinery (2Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar). The mammalian UPR is minimally composed of three transducers: Perk, Atf6, and Ire1 (5Schroder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2380) Google Scholar). Ire1 is highly conserved from yeast to mammals, but the homolog of Hac1 eluded scientists for many years. The mammalian counterpart of Hac1 was identified as Xbp-1. Xbp-1, a member of the CREB/ATF family of transcription factors, does not share any significant sequence homology with Hac1. It is composed of a basic leucine zipper-containing DNA binding domain located at the N terminus. The C terminus of Xbp-1 operates as a transcription activation domain. In contrast to yeast, in which Ire1p removes a relatively large intron from Hac1 mRNA, only 26 bases are excised by splicing the mRNA of Xbp-1. This splicing induces a frameshift in the coding sequence, replacing the 105-amino acid C terminus of the unspliced Xbp-1 protein with a 226-amino acid domain (6Yoshida H. Matsui T. Yamamoto A. Okada T. Mori K. Cell. 2001; 107: 881-891Abstract Full Text Full Text PDF PubMed Scopus (2869) Google Scholar, 7Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2066) Google Scholar). The spliced C terminus potently activates transcription of downstream target genes. Similar to yeast, the specific target genes of Xbp-1 encode proteins that enhance the folding capacity of the ER and participate in the clearance of misfolded proteins from the ER (8Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1564) Google Scholar). Despite high levels of the unspliced Hac1 mRNA, no unspliced Hac1 protein is detectable, caused by selective inhibition of its synthesis. The mechanism of the arrest in translation of Hac1 mRNA involves base pairing interactions between the intron and the 5′ untranslated region (9Ruegsegger U. Leber J.H. Walter P. Cell. 2001; 107: 103-114Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In contrast to Hac1, unspliced Xbp-1 (Xbp-1u) is continuously synthesized, but it is unstable and quickly degraded (10Lee A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9946-9951Crossref PubMed Scopus (494) Google Scholar). Once Xbp-1 mRNA is spliced, the encoded protein gains stability and allows activation of transcription. Lee et al. (10Lee A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9946-9951Crossref PubMed Scopus (494) Google Scholar) previously demonstrated that an N-terminal segment of Xbp-1, the domain shared between the unspliced and the spliced forms, is relatively stable and exerts dominant-negative activity for expression of genes activated by spliced Xbp-1 (Xbp-1s) targets. This is probably because of direct competition between the N-terminal segment of Xbp-1 and Xbp-1s for the available DNA binding sites. Furthermore, overexpression of a mutant of Xbp-1u, which cannot be spliced because of point mutations in the intron (referred to here as “unspliceable”), also displays dominant-negative characteristics, when assayed under ER stress conditions that robustly generate Xbp-1s. Finally, replacement of lysine residues with arginines at the C terminus of Xbp-1u increases its stability and potentiates its dominant negative activity. These observations implicate the ubiquitin-proteasome system as responsible for Xbp-1u degradation (10Lee A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9946-9951Crossref PubMed Scopus (494) Google Scholar). Overall, these data suggest that Xbp-1u is a potential inhibitor of the Xbp-1 pathway of the UPR, and its removal by proteolysis is a prerequisite for proper activation of the UPR. Here, we examined the localization and stability of Xbp-1u and its C terminus. We show that Xbp-1u is an intrinsically unstable protein, which is degraded in living cells at a rate comparable with its rate of synthesis. The C terminus of Xbp-1u is sufficient to mediate this exceedingly rapid degradation, which occurs predominantly in the cytoplasm involving ubiquitin-dependent and -independent pathways. Against expectation, improvement of the stability of Xbp-1u augmented the transcription of Xbp-1-specific and -nonspecific target genes under conditions that robustly induce the UPR. We conclude that the rapid degradation of Xbp-1u is required to prevent uncontrolled activation of the UPR. In Vitro Transcription and Translation—pcDNA3.1-encoded Xbp-1N terminus, Xbp-1u, and Xbp-1s were translated using the TnT coupled reticulocyte lysate system (Promega) according to manufacturer’s instructions, in the presence of 35Smethionine (PerkinElmer Life Sciences). To initiate the chase period we added RNase (1 mg/ml final concentration, Roche) together with excess unlabeled methionine. Equal aliquots were taken at the indicated time points, diluted in reducing sample buffer, boiled, and analyzed by SDS-PAGE (12%) followed by fluorography. Cell Lines—HeLa and Xbp-1-/- mouse embryonal fibroblasts (MEFs) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. ts20 cells were cultured at 32 °C in α-minimal essential medium supplemented with 10% fetal calf serum (11Kulka R.G. Raboy B. Schuster R. Parag H.A. Diamond G. Ciechanover A. Marcus M. J. Biol. Chem. 1988; 263: 15726-15731Abstract Full Text PDF PubMed Google Scholar). Epifluorescence Imaging—HeLa cells were seeded on glass coverslips 18 h before transfection. Vectors encoding GFP fused at its C terminus to the specified constructs were transfected using the calcium phosphate precipitation method (CalPhos, BD Bioscences). 24 h after transfection, nuclei were labeled with Hoechst 33342 (blue fluorescence, Invitrogen). Images were obtained with a Spot RT digital camera mounted on a TE300 Nikon microscope at ×40 magnification. Retrovirus Production—Wild type Xbp-1 and Xbp-1 mutants were cloned into the pMiG MSCV vector harboring an internal ribosomal entry site-GFP element to allow sorting of infected cells. Viral particles were made in 293T cells by triple transfection of the retroviral vector (2 μg), pMD-gag-pol (2 μg), and pVSV-G (2 μg) using Effectene (Qiagen). Cells were infected as previously described (12Iwakoshi N.N. Lee A.H. Glimcher L.H. Immunol. Rev. 2003; 194: 29-38Crossref PubMed Scopus (210) Google Scholar). Metabolic Labeling, Pulse-Chase Analysis, and Immunoprecipitation—Metabolic labeling, pulse-chase and were as described A. P. D. J. 2001; PubMed Scopus Google Scholar, B. Iwakoshi N.N. Lee A.H. Glimcher L.H. J. 2005; PubMed Scopus Google Scholar). was from was the GFP ts20 cells were transfected with that encodes an Xbp-1u using 24 when cells were to °C for was at 32 was with a was from using was synthesized from using the synthesis and green fluorescent were in an system The of were from the using as were by The were Xbp-1u an demonstrated rapid of Xbp-1u in M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. Nature. 2002; 415: 92-96Crossref PubMed Scopus (2066) Google Scholar, A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9946-9951Crossref PubMed Scopus (494) Google Scholar). To Xbp-1u is an intrinsically unstable protein, we examined its stability in a the TnT in translation we Xbp-1u, Xbp-1s, and the region of Xbp-1 shared between Xbp-1u and Xbp-1s, (referred to as This under The chase period was by of RNase and excess unlabeled into the in the N terminus of Xbp-1 was stable the chase period In Xbp-1u of the Xbp-1u was degraded the of the was stable the of the The of stable from to but of the amount of We that this Xbp-1u by means is from the degradation Xbp-1s more than Xbp-1u of as any on the degradation in not In we for the of polypeptides that might by of the the presence of polypeptides for Xbp-1u, in a of by that the reticulocyte lysate used for the of in translation is for These polypeptides time labeled by were in the of added to for proteins J. D. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We this for the N terminus of Xbp-1 for Xbp-1s and To that the which at than Xbp-1u, to modified by we translated Xbp-1u in the presence of The increases the of by and the of the We a in the of the high of Xbp-1u by The of Xbp-1u in the and its by that Xbp-1u degradation is a and well be The N and C of Xbp-1 and be into which are shared between XBP-1u and Xbp-1s, were as the Xbp-1N terminus. The at the of the intron acid and to the of Xbp-1u the segment referred to as is the domain to Xbp-1s, by removal of the short To the of the to localization and the of Xbp-1, we GFP for these Xbp-1u was used in a form that its splicing (referred to as as described (12Iwakoshi N.N. Lee A.H. Glimcher L.H. Immunol. Rev. 2003; 194: 29-38Crossref PubMed Scopus (210) Google Scholar). This was to prevent the splicing of mRNA by We also an mutant in which the located at and of the Xbp-1u, were by This referred to as the stability of Xbp-1u, probably by its (10Lee A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9946-9951Crossref PubMed Scopus (494) Google Scholar). cells were transfected with the GFP constructs and by The nucleus was by with Hoechst 33342 (blue In of the GFP proteins was by pulse-chase Xbp-1u no localization to the nucleus the cytoplasm and was in the The Xbp-1N terminus was to the because of the basic leucine domain and the three and was from the nucleus for not any located in the it be in that are and for as which are to We conclude that in the of the the the localization in trafficking of Xbp-1u in and out of the of the of the GFP proteins by pulse-chase rapid for and Cells were with 35Smethionine for of both and were degraded terminus stability and was at the chase In contrast to was for the in cells terminus was not stable and is sufficient to direct rapid degradation of an stable protein GFP, and because it localization to the cytoplasm, we conclude that the degradation of Xbp-1u occurs but not in the To the of Xbp-1u degradation, we fused the mutant of Xbp-1u to cells were transfected with its conditions of comparable transfection the mutant in its GFP fused form was more stable than the wild type as by pulse-chase at of of of the mutant of GFP in the inhibition of Xbp-1u degradation in in the cytoplasm, and that this is the of degradation. We a to Xbp-1s. Xbp-1s was predominantly to the nucleus of not show the to the cytoplasm, as was for was between the nucleus and the the localization of Xbp-1s is by its N-terminal for and indicated that also at a rate than unspliced We also that Xbp-1s more at the chase time in but not in with Xbp-1s is predominantly to the its and degradation probably To the rate at which Xbp-1u is degraded when at more we stable from in its The expression of these constructs was by a both proteins were by pulse-chase a inhibitor was the and the of the time at a chase time to the of the We also that of the inhibitor not these proteins from degradation, that activity of the not by is sufficient to degradation of Xbp-1u proteolysis as These that the rate of Xbp-1u degradation, by its C terminus, approaches its rate of synthesis. for the mechanism by which is we an of a protein that only the lysine residues at the C We also the in which these were by These constructs were transfected into 293T cells. was in the presence and of the inhibitor followed by polypeptides were by pulse-chase in the of a degradation rate to that of rate of synthesis both constructs the of improved stability of the the presence of in the sequence of of the C is not required for its degradation. the with it is when at N terminus T. H. J. J. H. PubMed Scopus Google Scholar). the degradation of might a but ubiquitin-independent degradation To this we a protein between and the by a quickly the between the and a this method polypeptides with the N-terminal of K. R. M. 2000; PubMed Scopus Google Scholar). The is to a C that no and a at its N terminus, and cannot be The and its mutant were transfected into 293T cells. was 24 h after transfection in the presence of the inhibitor We used of because is a inhibitor that be by in the of inhibitor of the was by C This degradation is probably by When in the presence of was and into that this is as in living cells. We this followed by a chase period of in the presence of to allow into Cells were to We followed the of its mutant from the protein in the presence of and degradation for both the and its of not the rate of degradation we not degradation of the we conclude that the removal of not the proteasomal the that the not any for was unstable and at comparable to the that in this degradation is The ts20 cells a activity. h at the is and the cells a arrest (11Kulka R.G. Raboy B. Schuster R. Parag H.A. Diamond G. Ciechanover A. Marcus M. J. Biol. Chem. 1988; 263: 15726-15731Abstract Full Text PDF PubMed Google Scholar). We used these cells to a for in the degradation of Xbp-1u was by transfection and the cells were for h at °C at 32 the The to °C the high on an internal control we followed the degradation of which is degraded by the in a ubiquitin-dependent A. A. M. S. J.S. M. F. Nature. PubMed Scopus Google Scholar). Xbp-1u was degraded at 32 °C we a in the rate of degradation, but degradation In degradation was at the the time used in the of Xbp-1u and We conclude that an ubiquitin-independent degradation mechanism to the overall elimination of of Xbp-1u the the unusually rapid degradation of Xbp-1u the mammalian UPR, we Xbp-1 mutants that Xbp-1u, but not Xbp-1s. The mutant was to a after the This as the stable N-terminal of the the mutant the of Xbp-1s We that the might impose conformational on the of the and might be required for as to be These interactions might a in degradation. We the with and to this Xbp-1 mutant as this was in a that does not the of Xbp-1s type Xbp-1 and the mutants were cloned into the retroviral harboring an internal ribosomal entry site-GFP Xbp-1-/- were with these was used as a negative GFP cells were and in of GFP were comparable for the control and the Xbp-1 constructs We assayed the expression and of Xbp-1 by pulse-chase type Xbp-1u was not inhibitor was These that Xbp-1u is degraded at a rate comparable with its rate of it from the mutant was and improved stability The mutant was at time but was of its degradation, but not it not We conclude that the the stability of Xbp-1u, it does not it from rapid degradation. To that the Xbp-1 constructs the of spliced, we the cells for h with these ER stress conditions, Xbp-1s was of Xbp-1s as a at with and degradation. these conditions, the synthesis of the mutant was that the synthesis of Xbp-1u and also but expression does not levels because of rapid degradation. we assayed expression of Xbp-1u with stability the of the UPR. To this we by the of mRNA of three target genes of the UPR in response to tunicamycin We the mRNA levels of a specific target of the Xbp-1 pathway (8Lee A.H. Iwakoshi N.N. Glimcher L.H. Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1564) Google a target shared by UPR J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. G. Lee Mol. Cell. Biol. 2005; PubMed Scopus Google and a target downstream to the pathway J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transcription was induced in Xbp-1-/- the GFP cells. with the Xbp-1 gene The of the Xbp-1-/- cells with the mutant the of in to with Xbp-1. This is probably because of the dominant negative activity of Xbp-1. We that cells the mutant were to Xbp-1 control cells with to of the Xbp-1 specific target Unexpectedly, were obtained when we examined the of and and We conclude that Xbp-1u, the overall of the UPR and expression not only of Xbp-1-specific but also of genes not Xbp-1 targets. proteins are a to control protein levels for when by the is degraded with a of P. R. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are for degradation by the proteins and also with a of L. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. J. Nature. PubMed Scopus Google Scholar). of these degradation are by pulse-chase Here we a degradation when in stable under the control of as the Xbp-1u is not by We also the degradation of Xbp-1u in a and in reticulocyte We that this rate of is not by the C terminus of the unspliced and the for The is that no levels of Xbp-1u are of degradation are on was demonstrated to and the degradation of and G. J. L. C. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, G. P. C. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of activity and the degradation of both by a ubiquitin-independent for this mechanism occurs of its ubiquitin-dependent We that to involving both ubiquitin-dependent and -independent to the degradation of cells a mechanism to expression of unspliced Hac1p, the counterpart of Xbp-1. This is caused by a in translation to prevent the expression of unspliced Hac1p the expression of the unspliced Xbp-1 and Hac1 proteins be We that Xbp-1u, UPR To this we Xbp-1 in which only the unspliced was in its the C domain. This mutant a stable Xbp-1 that to the nucleus and with Xbp-1s, and so in a dominant negative The was to the of Xbp-1u, consequently its interactions with the degradation and its degradation. This was only but a more stable We analyzed the expression of these constructs in Xbp-1-/- to to the UPR. We the synthesis of the Xbp-1 mutant even under conditions of ER induced by tunicamycin This that under conditions Xbp-1u is synthesized under of Xbp-1 as in the of into in the of the of the mRNA of Xbp-1, in its unspliced It that Xbp-1s and Xbp-1u comparable at any time Xbp-1u expression which might for a to by of the UPR. The Xbp-1 mutants to this When we analyzed the of UPR target genes in reconstituted with the of Xbp-1, we were to of and in the mutant as with We cannot the that the is a of we not of when was by in an We also analyzed the localization of a Xbp-1 protein, and found it between the nucleus and cytoplasm, the must with components of the UPR signaling in the of activation by tunicamycin of these constructs in the activated UPR targets upon tunicamycin and H. L. the of Xbp-1u to the overall activation of UPR targets is in the of Xbp-1u for levels of transcription of genes was by et al. K. Kaufman R.J. 2005; PubMed Scopus Google Scholar). In genes the UPR machinery for transcription these required required genes between the C. The between and be to the transcription capacity of in C. Xbp-1u for the transcription of the of the UPR targets K. Kaufman R.J. 2005; PubMed Scopus Google Scholar). We conclude that under UPR conditions, the presence of levels of Xbp-1u the UPR signaling The of with Xbp-1u degradation in the of a living to be