|
|
|
|
|
|
|
||
Article |
Correspondence to Yukio Kimata: kimata{at}zero.ad.jp; or Kenji Kohno: kkouno{at}bs.naist.jp
Chaperone protein BiP binds to Ire1 and dissociates in response to endoplasmic reticulum (ER) stress. However, it remains unclear how the signal transducer Ire1 senses ER stress and is subsequently activated. The crystal structure of the core stress-sensing region (CSSR) of yeast Ire1 luminal domain led to the controversial suggestion that the molecule can bind to unfolded proteins. We demonstrate that, upon ER stress, Ire1 clusters and actually interacts with unfolded proteins. Ire1 mutations that affect these phenomena reveal that Ire1 is activated via two steps, both of which are ER stress regulated, albeit in different ways. In the first step, BiP dissociation from Ire1 leads to its cluster formation. In the second step, direct interaction of unfolded proteins with the CSSR orients the cytosolic effector domains of clustered Ire1 molecules.
 |
Introduction |
|---|
 TopAbstract Introduction ResultsDiscussionMaterials and methodsReferences |
|---|
and IREß, as well as a second type I transmembrane protein, pancreatic ER kinase (PERK), which attenuates protein translation by phosphorylation of eukaryotic translation initiation factor 2 (Harding et al., 1999). A third system is the membrane-anchored transcription factor ATF6. Upon ER stress, ATF6 is solubilized by proteolysis and up-regulates ER chaperone and other genes (Haze et al., 1999). Because the luminal domains of Ire1 and PERK show moderate amino acid sequence similarity, we believe that they sense ER stress by the same mechanism. One breakthrough finding toward understanding the ER stress-sensing mechanism was the observation that BiP binds to Ire1 and dissociates in response to ER stress (Bertolotti et al., 2000; Okamura et al., 2000). It is highly likely that BiP binding negatively regulates Ire1 because activity of Ire1 is considerably attenuated in yeast BiP mutants in which dissociation of the mutated BiP proteins from Ire1 is impaired (Kimata et al., 2003, 2004). However, as described in the next paragraph, dissociation of BiP from Ire1 is not sufficient for activation of Ire1.
We previously predicted that the luminal domain of yeast Ire1 is composed of five subregions (I–V; Fig. 1).
This is because our systematic mutagenesis analysis demonstrated that 10 aa deletions in subregions II and IV, but not in subregions I, III, or V, inactivate Ire1 (Kimata et al., 2004). Our speculation that subregions I and V are loosely folded is supported by the finding that these subregions are highly sensitive to proteolysis in the context of a recombinant Ire1 luminal domain protein (Oikawa et al., 2005). According to the crystal structure presented by Credle et al. (2005), subregions II–IV form one tightly folded domain, which we termed the core stress-sensing region (CSSR). Both this crystal structure analysis and our systematic mutational analysis suggest that subregion III exists as a flexible stretch sticking out from the CSSR. The BiP binding site is located in subregion V (Kimata et al., 2004). Importantly, an Ire1 mutant that contains a deletion of almost all of subregion V (hereafter called 
V; see Fig. 1 for the position) is not constitutively active, but is regulated by ER stress as well as wild-type Ire1, even though BiP does not bind to this mutant (Kimata et al., 2004).
|
luminal domain, Zhou et al. (2006) argued that this groove is not suited to capture unfolded proteins. Moreover, no biochemical evidence has been provided for direct binding of the CSSR to unfolded proteins.
The oligomerization status of Ire1 is also enigmatic (Kohno, 2007). Epitope-tagged Ire1 coimmunoprecipitates with differently epitope-tagged Ire1 from lysate of ER-stressed cells, but only to a small extent from that of nonstressed cells (Kimata et al., 2004). We reported recently, from coimmunoprecipitation analysis, that Ire1 seems to be fully self-associated, even in the absence of ER stress, when it carries both the V mutation and another deletion of the N-terminal three-quarters of subregion I (hereafter called I; see Fig. 1 for the position; Oikawa et al., 2007). This finding suggests that the self-association is regulated both by subregion V (probably by binding and dissociation of BiP) and subregion I. Notably, activation of this IV mutant Ire1 was still dependent on ER stress (although IV Ire1, whose luminal domain consists of almost only the CSSR, was named "core mutant" in our previous paper, we do not use this name in the present paper because it can be confused with "a mutation in the CSSR"; Oikawa et al., 2007). According to density gradient fractionation of cell lysates, it seems that the oligomerization status of Ire1, upon activation by ER stress or when carrying the IV mutation, is dimeric (Bertolotti et al., 2000; Oikawa et al., 2007). Furthermore, recombinant Ire1 luminal domain or CSSR exists in dimeric form in solution (Liu et al., 2002; Oikawa et al., 2005). However, the crystal structure suggests that the CSSR forms higher order oligomers because one CSSR molecule is in contact with two CSSR molecules via different interfaces (Credle et al., 2005). The authors argue that this higher order oligomerization is crucial for activation of Ire1 because point mutations that are deduced to deform either interface considerably weaken Ire1 activity. Nevertheless, formation of such higher order oligomers in vivo has not been demonstrated.
One scenario proposed by Credle et al. (2005) and modified by us (Oikawa et al., 2007) explains these confusing observations. In this explanation, a combination of BiP dissociation and release from a negative regulation by subregion I lead Ire1 to dimerize. Unfolded proteins bind and may tether dimerized CSSRs, which results in highly oligomerized and active Ire1. Because the higher order oligomer is unstable, it cannot be detected in cell lysates or in solutions of recombinant Ire1 fragments. In the present study, we provide some lines of evidence that in vivo Ire1 actually forms higher order oligomers, here called clusters, and interacts with unfolded proteins. Furthermore, we describe a new scenario in which these two events are positioned differently from the aforementioned scenario.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
IV deletion and Ser103 to Pro (S103P) point mutations (see Fig. 1 for the positions and its legend for the amino acid numbering; Oikawa et al., 2007). Here, we checked cellular localization of Ire1 and its mutants by immunofluorescent staining of C-terminally HA-tagged molecules. As shown in Fig. 2 A, HA- tagged wild-type Ire1 (Ire1-HA) demonstrated an ER-like staining pattern in nonstressed cells, whereas the S103PIV mutation changed localization of Ire1-HA to a clumped distribution.
A dotlike distribution, hereafter called clusters, was also observed in wild-type Ire1-HA cells exposed to ER stress by treatment with tunicamycin (Tun) or DTT, although there was also residual ER-like distribution (Fig. 2, B and C). To our knowledge, this paper is the first demonstration of a localization change of Ire1.
|
IV Ire1-HA in nonstressed cells and of wild-type Ire1-HA in ER-stressed cells was observed even with expression from centromeric plasmids (Fig. 2 D, S103PIV and WT [Tun]), although this fluorescent signal was faint. This finding indicates that the cluster formation is not an artifact caused by high expression from multicopy plasmids. Furthermore, we constructed an Ire1-HA gene knockin strain, here named YKY2005. Western blot detection of Ire1-HA showed that the expression level of Ire1-HA in YKY2005 cells is slightly higher than that in ire1 cells in which Ire1-HA is expressed from a centromeric plasmid (Fig. S2). As expected, clusters of Ire1-HA were observed in YKY2005 cells when they were exposed to ER stress (Fig. 2 E). Clustered Ire1-HA in DTT-treated cells quickly reverted to the normal ER-like distribution when the cells were incubated under nonstressed conditions (Fig. 2 F). This observation suggests that the cluster is not a simple aggregate of the protein.
Cluster formation of S103PIV Ire1-HA was observed even in cells carrying the hac1 mutation (Fig. 2 G) or when a kinase-inactive mutation, K702A, was introduced (Fig. 2 H). Thus, activation of either Ire1 or the UPR signaling pathway (Ire1-HAC1 pathway) is not a prerequisite for cluster formation. For cluster formation, the combination of the I and V mutations was necessary and sufficient, whereas the S103P mutation did not contribute (Fig. 2 I). Total cellular amount of Ire1-HA did not change considerably upon introduction of any of these mutations (Oikawa et al., 2007), and thus expression level is not an important determinant of the localization.
As presented in electron micrographs (Fig. 3 A), cells with multicopy expression of S103PIV Ire1-HA often showed abnormally folded cisternae that were probably derived from the ER.
Importantly, immunogold labeling of anti-HA antibody demonstrated that the epitope was located on these membranous structures. These observations strongly suggest that Ire1 clusters on the membrane of the ER, which is deformed by the cluster formation. When the cluster is smaller, as in the case of single copy expression of Ire1-HA, the immunogold signals of the Ire1-HA clusters and the abnormal membranous structures are likely to be more difficult to find. Thus it is unclear whether cluster formation of endogenous Ire1 under ER-stress conditions leads to such morphological changes of the ER.
|
IV Ire1-HA and ER marker protein GFP-Sec12 (Sato et al., 2003) were doubly stained with anti-HA and anti-GFP antibodies and observed using an ApoTome-based optical sectioning system. Most of the clusters (>90%) localized on or touched the anti-GFP–stained area, suggesting again that the clusters are formed in the ER. It is likely that not all of the clusters exactly localize on the anti-GFP–stained area because they may exclude other membrane proteins including GFP-Sec12. As shown in Fig. 3 D, the clusters did not colocalize with Golgi marker protein Mnn9.
Cluster formation of Ire1 is caused by two different types of homomeric interaction of the CSSR
According to the crystal structure (Credle et al., 2005), two CSSR molecules contact via two different interfaces (I and II; see Fig. 8), which suggests a possibility for formation of higher order oligomers. The crystal structure also predicts that the point mutations of Ire1, F247A and W426A (see Fig. 1 for the positions), respectively deform interfaces I and II. As shown in Fig. 4 A, both the F247A and the W426A mutations abolished cluster formation, although F247A Ire1-HA clustered in 
20% of total cells treated with Tun.
This finding strongly suggests that the cluster formation is caused by the high order oligomerization of the CSSR.
|
IV Ire1-HA is coexpressed with a C-terminally FLAG-tagged version (Ire1-FLAG) of the same mutant, the FLAG-tagged version is efficiently coimmunoprecipitated with the IV Ire1-HA from the cell lysate even in the absence of ER stress (Fig. 4 B; Oikawa et al., 2007). The F247A mutation, but not the W426A mutation, considerably reduced the level of coimmunoprecipitation (Fig. 4 B). This finding suggests low affinity of the association via interface II.
In Fig. S3 (available at http://www.jcb.org/cgi/content/jcb.200704166/DC1), cells were treated with the noncleavable cross-linking reagent disuccinimidyl suberate, and the lysate was analyzed by anti-HA Western blotting. The amount of IV Ire1-HA migrating to the original position was significantly decreased by treatment of cells with disuccinimidyl suberate, whereas wild-type Ire1-HA was less affected. The smear signal produced by high molecular mass protein is more intense in lane 4 than in lane 2 (Fig. S3), although highly cross-linked proteins may be harder to detect for several reasons, including extremely low mobility in the gel. Altogether, this finding supports highly efficient cross-linking and thus high order oligomerization of IV Ire1-HA.
Cluster formation is necessary but not sufficient for activation of Ire1
We then monitored activity of the Ire1 mutants by using a UPR element (UPRE)-reporter construct, from which expression of ß-galactosidase is driven by a UPRE (Mori et al., 1992; Kohno et al., 1993). The activity of I Ire1 and V Ire1 was as tightly regulated as that of wild-type Ire1 (Fig. 5 A, 2–4), whereas IV Ire1 was slightly activated even in nonstressed cells (Fig. 5 A, 5; Oikawa et al. 2007).
Because extrinsic ER stress was required, even for full activation of IV Ire1, which was constitutively clustered, we think that cluster formation is not sufficient for full activation of Ire1.
|
IV Ire1 (Fig. 5 B, 3 and 4) or wild-type Ire1 (Credle et al. 2005), the activity was considerably compromised. This finding strongly suggests that cluster formation is a prerequisite of Ire1 activation. Although the W426A mutation has a stronger negative effect on activity and cluster formation than the F247A mutation (Figs. 4 A and 5 B), coimmunoprecipitation of Ire1-FLAG with Ire1-HA was considerably impaired only by the F247A mutation (Fig. 4 B), suggesting again that a kind of homomeric association of Ire1 is not detected by the coimmunoprecipitation analysis.
A deletion of subregion III (hereafter called III; see Fig. 1 for position) significantly reduced activity of wild-type and IV Ire1 (Fig. 5 A, 6 and 7). This finding was further confirmed by directly checking splicing of HAC1 mRNA (Fig. 5 F, lane 10). It should be noted that the III mutation did not impair cluster formation either by extrinsic ER stress or by the IV mutation (Fig. 6 A), nor did it reduce cellular expression level of Ire1 (Fig. 6 B).
These observations strongly suggest that in addition to cluster formation (see Step 1 in Fig. 8), the CSSR is responsible for another step, hereafter called Step 2 (see Fig. 8), which is also necessary for Ire1 activation.
|
IV Ire1 was mutagenized such that it had 4 aa serial deletions, from aa 527–530 to 567–570 (see Fig. 1 for the positions), and was subjected to the UPRE-lacZ reporter assay, which showed that none of these mutations inactivate Ire1 (Fig. 5 C). On the contrary, IV Ire1 was activated by the deletion of 4 aa residues, LLSK, located at the cytosolic end of the transmembrane domain even in the absence of ER stress (Fig. 5 C, 12; and see Fig. 1 for the position). This mutation, hereafter called LLSK, did not significantly alter activity of wild-type Ire1 (Fig. 5 D [6] and F[lanes 6 and 13]). However, the impaired- activation phenotype of the III mutation was suppressed by the LLSK mutation (Fig. 5 D [7] and F [compare lane 14 to 10]). Shift of mobility on SDS-PAGE by Endo H digestion indicates that LLSK Ire1, as well as wild-type Ire1, is N-glycosylated (Fig. 6 C). We also checked whether wild-type and LLSK Ire1 could be extracted from the yeast microsome fraction by various reagents (Fig. 6 D). Although both of the Ire1 variants were resistant to extraction with sodium chloride and sodium carbonate, they were partially extracted by Triton X-100. Together with ER localization (Fig. 6 A, bottom left) and activity of LLSK Ire1, these observations show that the LLSK mutation does not prevent Ire1 from being an ER-located type I transmembrane protein, although the LLSK residues may be a part of the transmembrane domain. Cluster formation of Ire1 was not affected by the LLSK mutation (Fig. 6 A). Collectively, our results demonstrate that the LLSK mutation abolishes the requirement of Step 2 for activation of Ire1, but not the requirement of the cluster formation.
S103P, as well as LLSK, is likely to be a mutation that does not facilitate cluster formation (Fig. 2 I) but which activates clustered Ire1. The S103P mutation confers a constitutive-activation phenotype on IV Ire1 (Fig. 5 E, 2; Oikawa et al., 2007) but not on wild-type Ire1 (Fig. 5 D, 4). Also, the S103P mutation suppressed the impaired-activation phenotype of the III mutation (Figs. 5 D [5] and F [compare lane 12 to 10]).
It is notable that the constitutive-activation phenotypes of both S103PIV Ire1 and IVLLSK Ire1 were only moderately compromised by the III mutation, whereas IIIIV Ire1 carrying neither the S103P nor the LLSK mutation was almost completely inactive (Fig. 5 E, compare 3 to 2). In contrast, the clustering-impaired mutations F247A and W426A almost completely attenuated basal activity of IV Ire1, even when carrying the S103P or the LLSK mutation (Fig. 5 E, 4 and 5).
Fig. 6 E shows the results of a coimmunoprecipitation experiment that probed the physical interaction between Ire1 variants and BiP. As described in our previous papers (Okamura et al., 2000; Kimata et al., 2003, 2004; Oikawa et al., 2007), Ire1-HA or mutants were expressed from 2-µm plasmids, and the cells were lysed and subjected to anti-HA immunoprecipitation. Double bands of Ire1-HA in the top and third panels (Fig. 6 E), which were also shown in Figs. 6 D, S2, and S3, are caused by partial degradation in subregion I (Oikawa et al., 2007). Anti-BiP Western blotting of the anti-HA immunoprecipitates (Fig. 6 E, bottom) indicates BiP binding to Ire1-HA and its dissociation upon ER stress, neither of which were affected by either the III or the LLSK mutation. S103P Ire1-HA also shows BiP binding and dissociation at similar levels to wild-type Ire1-HA (Oikawa et al., 2007).
The CSSR interacts directly with unfolded proteins
Finally, the possibility that the CSSR binds directly to unfolded proteins was explored by monitoring its ability to inhibit aggregation of denatured proteins in vitro. For this experiment, the CSSR was bacterially expressed as an N-terminally maltose binding protein (MBP)–fused and C-terminally His8-tagged recombinant protein, hereafter called MBP-CSSR. Integrity and purity of wild-type and mutant MBP-CSSRs and an unfused MBP control were verified by SDS-PAGE analysis (Fig. 7 A).
Firefly luciferase and porcine citrate synthase were denatured by guanidine HCl, and then the aggregation induced by dilution of the denaturing mixtures was monitored by measuring the increase of turbidity. Fig. 7 (B and C) shows that aggregation of either denatured protein was not attenuated by the unfused MBP control, whereas MBP-CSSR showed significant effects in inhibiting the aggregation. This finding indicates that the CSSR can interact with two models of aggregation-prone substrates, which suggests that this domain binds to unfolded regions on polypeptides.
|
III mutation as an activation-impaired mutation. This is because, as shown in Fig. 5, the phenotype of the III mutation was suppressed either by the S103P or the LLSK mutation, which indicates that the global structure of Ire1 is not perturbed by the III mutation. As shown in Fig. 7 (B and C), the III mutant version of MBP-CSSR did not attenuate aggregation of the denatured proteins.
Does the groove-like structure described in the Introduction contribute to interaction of the CSSR with unfolded proteins? We modified MBP-CSSR to carry combined substitutions of 3 aa residues (M229A/F285A/Y301A) facing into the groove, which abolish activation of Ire1 (Credle et al., 2005). This mutant MBP-CSSR did not inhibit aggregation of unfolded proteins (Fig. 7, B and C), supporting the idea that unfolded proteins are captured by the groove. Nevertheless, it is likely that the M229A/F285A/Y301A mutation confers more extensive damage to the CSSR than the III mutation, which may include perturbation of global protein structure, because the impaired-activation phenotype of M229A/F285A/Y301A Ire1 was not rescued either by the S103P or the LLSK mutation (Fig. 5 E, 6).
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
cells and because activity of Ire1 was impaired by either the F247A or the W426A mutation. Clusters of wild-type Ire1 quickly dissociated upon removal of ER stress. This finding again implies that the cluster formation is biologically relevant. The molecular mechanism by which this cluster dissociation is promoted is currently unclear. The cytosolic domain of yeast Ire1 carries a highly basic sequence, which, according to Goffin et al. (2006), acts as an NLS when fused with other proteins. However, our immunofluorescent analysis indicated that both unclustered and clustered Ire1 variants are distributed not only at the nuclear rim but also at other parts of the ER. We thus think that this highly basic sequence does not function as an NLS in the authentic Ire1 molecule.
Another feature of the CSSR that is predicted by the crystal structure but for which there is no supporting biochemical evidence is its direct binding to unfolded proteins (Credle et al. 2005). Here, we also demonstrate that the CSSR actually interacts with unfolded proteins by monitoring its ability to inhibit aggregation of denatured proteins in vitro. Because this property of the CSSR was abolished by the III mutation, we think that this interaction is biologically meaningful and required for activation of Ire1. The result from the M229A/F285A/Y301A mutation supports the idea that unfolded proteins are captured by the groove-like structure of the CSSR. We failed to demonstrate in vivo interaction between unfolded model proteins and Ire1 by coimmunoprecipitation, even from cells treated with a chemical cross-linker, dithiobis succinimidyl propionate. The interaction may be weak and transient, and in addition, we speculate that because of a structural reason, the cross-linking between the proteins is inefficient.
Unlike a previous scenario detailed in the last paragraph of the Introduction, we now believe that there exists a regulatory step, called here Step 2, other than the cluster formation. This is because IV Ire1 was constitutively clustered, but extrinsic ER stress was still required for full activation of this mutant. Furthermore, the S103P and the LLSK mutations are likely to abolish the requirement of Step 2 for activation of Ire1; in contrast, the III mutation impairs progression of Step 2 but not of the cluster formation. Importantly, such phenotypes of these mutations support our proposal about requirement of Step 2 for activation of Ire1.
What triggers cluster formation or Step 2? Unlike IV Ire1, either I or V single mutant showed normal ER-like localization. This finding indicates that the cluster formation requires both dissociation of BiP or the BiP-nonbinding mutation V, and release from repression by subregion I. The mechanism by which subregion I negatively regulates Ire1 remains unclear. Considering BiP's ability to recognize a wide variety of unfolded proteins, it is an attractive idea that BiP acts as a sensor for unfolded proteins in the cluster-formation step, although Ire1 may positively contribute to its own dissociation from BiP (Kimata et al., 2004). In contrast, BiP is not involved in Step 2 because the BiP-nonbinding mutant IV Ire1 undergoes regulation in Step 2 and because none of the Step 2 mutants (III, S103P, or LLSK) affect BiP binding and its dissociation from Ire1. Impairment of the interaction between denatured proteins and the CSSR protein carrying the III mutation strongly suggests that Step 2 is regulated by direct interaction of unfolded proteins with Ire1. Considering the constitutive cluster formation of IV Ire1, we think that direct interaction of unfolded proteins is not required in Step 1.
Another important question is what change is produced in Step 2. Because the LLSK mutation, which is located at the cytosolic end of the transmembrane domain, abolishes the requirement of Step 2 for full activation of Ire1, it is likely that orientation of the cytosolic domain is tightly related to Step 2. We propose that, as illustrated in Fig. 8, physical interaction of unfolded proteins with the CSSR causes conformational change of the luminal domain, which leads to reorientation of the cytosolic domain, without changing oligomerization status.
This proposal is similar to the case for some cytokine receptors, which, upon binding of ligands, undergo not only self-association but also conformational change, causing alteration of cytosolic-domain orientation.
|
V Ire1 (Kimata et al., 2004).
Nevertheless, it is likely that not all events noted in Fig. 8 are required to obtain partial activation of Ire1. Indeed, the constitutively clustering mutant IV Ire1 is slightly but significantly activated even without extrinsic ER stress. Furthermore, Liu et al. (2000) reported substantial activation of chimeric Ire1 mutants in which the luminal domain was replaced by dimer-forming fragments of transcription factor proteins. More recently, Lipson et al. (2006) reported that transient activation of mammalian IRE1 in pancreatic ß cells exposed to high levels of glucose does not accompany BiP dissociation.
Is the mechanism presented here applicable to Ire1 orthologues? Because mammalian and plant orthologues of Ire1 do not carry regions corresponding to subregion I, their self-association may be regulated solely by BiP. It should be noted that Zhou et al. (2006) reported a crystal structure of the luminal domain of mammalian IRE1, which suggests that neither high order oligomerization nor direct binding of unfolded protein is likely. Unlike cells of the unicellular organism yeast, which suffer direct environmental stress and carry only one known ER-stress sensor Ire1, mammalian cells live in sophisticatedly regulated conditions and have more complicated pathways to respond to ER stress. Thus, it is not unreasonable to postulate that the regulatory mechanisms of the mammalian ER-stress sensors are different from those of yeast Ire1. Nevertheless, we do not think that mammalian IRE1 is regulated solely by BiP because a mutant of this protein with a deletion of the entire region corresponding to subregion V was still regulated by ER stress (unpublished data).
Whereas PERK's activity is apparently regulated by binding and dissociation of BiP (Bertolotti et al., 2000; Ma et al., 2002), a recombinant fragment of its luminal domain has been shown to inhibit aggregation of unfolded proteins in vitro (Yohda, M. et al. 2006. Proceedings of the 20th International Union of Biochemistry and Molecular Biology Congress and the 11th Federation of Asian and Oceanian Biochemists and Molecular Biologists Congress). Therefore, it is likely that in a similar manner to yeast Ire1, PERK is regulated both by BiP and by direct binding of unfolded proteins. Interestingly, ATF6 may also be regulated dually in its activation upon ER stress, although it has no structural similarity to Ire1 or PERK. Activation (i.e., transport to Golgi apparatus) of ATF6 is negatively regulated by binding of BiP and by intra- and intermolecular disulfide bridge formation, both of which are lowered upon ER stress (Shen et al., 2002; Nadanaka et al., 2007). Finally, such multiplicity of regulatory mechanisms implies complexity of conditions in which these ER-stress sensors are individually activated, as suggested by DuRose et al. (2006).
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
null mutation, were used according to the difference of their mating and auxotrophic phenotypes. To generate the ire1 hac1 strain YKY1004, a EUROSCARF strain Y15650 (MAT ura3 leu2 his3 lys2 hac1::kanMX4; provided by J.W. Goethe, University Frankfurt, Frankfurt, Germany) was modified to carry a complete deletion of the IRE1 ORF by replacement with the URA3 gene. To obtain a YKY2005 strain, the Ire1-HA gene was knocked in to replace the endogeneous IRE1 gene of wild-type strain KMY1005 (MAT ura3 leu2 his3 trp1 lys2), which is congenic to KMY1015, KMY1516, and KMY1520. Cells were exponentially cultured in SD medium (2% glucose and 0.66% yeast nitrogen base without amino acids; Difco), supplemented with appropriate nutrients, at 30°C. In our previous studies (Okamura et al., 2000; Kimata et al., 2003, 2004), plasmids pRS313-IRE1, pRS315-IRE1-HA, pRS423-IRE1-HA, and pRS426-IRE1-FLAG were generated by insertion of the IRE1 gene (or its C-terminally epitope-tagged version) into yeast centromeric vectors pRS313 and pRS315 (Sikorski and Hieter, 1989) and 2-µm vectors pRS423 and pRS426, respectively (Christianson et al., 1992). As described in Kimata et al. (2004), we introduced IRE1 mutations into these plasmids by in vivo homologous recombination (gap repair) between the IRE1 plasmids cleaved by restriction enzymes and IRE1 mutant fragments created by overlap PCR, primers for which are listed in Table S1 (available at http://www.jcb.org/cgi/content/jcb.200704166/DC1). A UPRE-lacZ reporter plasmid pCZY1 (URA3 2 µm) was provided by K. Mori (Kyoto University, Kyoto, Japan). A GFP-Sec12 expression plasmid pSKY54-GFP-SEC12 was provided by A. Nakano (Institute of Physical and Chemical Research, Saitama, Japan). A pRS315-based derivative of pSKY54-GFP-SEC12 was generated by in vivo homologous recombination between PvuII-digested pSKY54-GFP-SEC12 and XhoI–NotI–digested pRS315.
Fluorescent microscopy
For fluorescent microscopic examination, cells were fixed in 0.1 M potassium acetate buffer, pH 6.8, containing 3.3% formaldehyde for 2 h and processed according to Oka et al. (1998). Antibodies used are listed in Table S2 (available at http://www.jcb.org/cgi/content/jcb.200704166/DC1), including anti-Mnn9 antiserum, which was provided by Y. Noda (The University of Tokyo, Tokyo, Japan). The mounting medium was 90% glycerol containing 0.1% p-phenylenediamine. For conventional fluorescent microscopy, Axiophoto (Carl Zeiss MicroImaging, Inc.) was used with an oil immersion lens (Plan-Neofluor 100/1.30), and images were captured by a digital charge-coupled device (CCD) camera system (DP70; Olympus) carrying built-in software for image acquisition. For deconvolution microscopy, an Axiovert 200M (100/1.40 oil immersion Plan Apochromat objective; Carl Zeiss MicroImaging, Inc.) with the Apotome system was used. Unless noted, FITC was used as fluorochrome. Photoshop software (Adobe) was used for conversion to grayscale images (Figs. 2, 4 A, and 6 A) and image overlapping (Fig. 3 B).
Electron microscopy
Cells were fixed basically as described previously in Sun et al. (1992). Cells were frozen in a high pressure freezer (HPM010; Bal-Tec Inc.) and transferred to anhydrous acetone containing 2% OsO4 in an automatic freeze-substitution apparatus (EM AFS; Leica) in which the temperature was gradually sifted from –80 to 23°C. After washing three times with anhydrous acetone, the samples were infiltrated with increasing concentrations of Spurr's resin in anhydrous acetone, and finally with 100% Spurr's resin. After polymerization in capsules at 60°C, ultrathin sections were cut on a microtome (Ultracut UCT; Leica). The sections were immunostained with anti-HA antibody and 10 nm of gold immunogold conjugate EM goat anti–mouse IgG (Table S2) and stained with 3% uranyl acetate for 2 h. The sections were then examined with an electron microscope (H-7600; Hitachi) at 100 kV.
RNA and protein analyses of yeast cells
RNA extraction, Northern blotting, cell lysis for protein analyses, immunoprecipitation, and Western blotting were performed as described previously (Kimata et al., 2003, 2004). Detergent-free cell lysates used to obtain microsome fractions were prepared as described in Craven et al. (1996). Radioactive signal from Northern blots was detected using a phosphor imager (BAS-2500; Fuji). For SDS-PAGE, lysates from 107 cells and IPs from 3 x 107 cells were run on precast gels (Multigel II Mini; Daiichi Pure Chemicals; 7.5% acrylamide), unless otherwise noted. The chemiluminescent signal from Western blots was captured by a cooled CCD camera system (LAS-1000plus; Fuji) and quantified using imaging software (ImageGauge; Fuji). When obtaining the Ire1-FLAG/Ire1-HA values in Fig. 4 B, we confirmed the linear relation between the quantified chemiluminescent signal and the actual amount of the band protein. Antibodies used, including that against yeast BiP (Higashio et al., 2000), are listed in Table S2.
Bacterial expression and purification of MBP-CSSR and its mutant variants
To avoid artifactual disulfide bond formation, all versions of MBP-CSSR had all Cys residues changed to Ser because this amino acid replacement does not affect activity of Ire1 (Oikawa et al., 2005). An IRE1 gene partial fragment corresponding to the CSSR was PCR amplified from pRS315-IRE1(CS)-HA (Oikawa et al., 2005) using primer set P-5 and P-6, digested with BamHI and HindIII, and inserted into similarly digested pMAL-c2x (New England BioLabs, Inc.). To generate mutant variants, mutations were introduced by using the overlap PCR mutagenesis technique as described in Kimata et al. (2004). The PCR primers are listed in Table S1.
An Escherichia coli strain BL21 codon plus (DE3)–RIL (Strategene) was transformed with one of the resulting plasmids and cultured at 37°C in 400 ml of 2x YT medium. Expression of MBP-CSSR or its mutant variants was induced by addition of IPTG (0.3 mM final concentration) into the culture, followed by further incubation at 30°C for 1 h. After harvest, cells were suspended in 15 ml of E. coli lysis buffer (50 mM Hepes, pH 8.0, 300 mM KCl, 5 mM MgCl2, 10 mM imidazole, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 0.4 mg/ml benzamidine, 0.4 mg/ml pepstatin A, 0.4 mg/ml leupeptin, 0.3 mg/ml lysozyme, and 14 U/ml DNase I; Takara) and disrupted by ultrasonication. The lysate was clarified by centrifugation (SRX-201; Tomy; 8,200 rpm for 10 min) and incubated with 0.5 ml of HisLink protein purification resin beads (Promega) for 12 h. The beads were packed into an 8-mm-diam column and sequentially washed with 6 ml of 50 mM Hepes, pH 8.0, 1 M KCl, 5 mM MgCl2, and 0.1% Triton-X 100; 6 ml of 50 mM Hepes, 300 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, and 20 mM imidazole; 6 ml of 50 mM Hepes, 300 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, and 40 mM imidazole; 3 ml of 50 mM Hepes, 300 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, and 60 mM imidazole; 5 ml of 50 mM Hepes, 300 mM KCl, 5 mM MgCl2, and 10 mM ATP; and 3 ml of 20 mM Hepes, 100 mM KCl, 5 mM MgCl2, and 50% (vol/vol) glycerol. Bead-bound proteins were eluted with 50 mM Hepes, 100 mM KCl, 5 mM MgCl2, 200 mM imidazole, and 50% (vol/vol) glycerol, and elution fractions (1 ml each fraction) were analyzed by SDS-PAGE.
In vitro protein aggregation assay
Citrate synthase (Roche) was dialyzed against 20 mM Hepes, pH 7.0, 150 mM KCl, 2 mM MgCl2, and 10% (vol/vol) glycerol, and stored at –80°C. Luciferase (25 µM final concentration) or citrate synthase (50 µM final concentration) were denatured by incubation in guanidine HCl– denaturing solution (guanidine HCl [6 M for luciferase or 4 M for citrate synthase], 20 mM Hepes, 50 mM KCl, and 2 mM MgCl2) for 30 min at room temperature. The denaturing mixture was then diluted with assay buffer (20 mM Hepes, pH 7.2, 50 mM KCl, and 2 mM MgCl2) in the presence or absence of MBP-CSSR (wild type or mutant) or MBP, and aggregate formation was monitored by absorbance at 320 nm with a spectrophotometer (DU640; Beckman Coulter) at room temperature.
Online supplemental material
Table S1 lists Ire1 mutations analyzed in this study. PCR primers used for generation of these mutations are also listed. Table S2 lists antibodies used in this study. Fig. S1 shows that even when expressed from a multicopy plasmid, Ire1-HA was activated in an ER stress-dependent manner. In Fig. S2, Ire1-HA was expressed from a single copy plasmid, a multicopy plasmid, and the knocked in gene, and the expression levels were compared. Fig. S3 shows highly efficient intermolecular homo-cross-linking of IV Ire1-HA, which supports high order oligomerization of this molecule. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200704166/DC1.
|
Acknowledgments |
|---|
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (14037240 to K. Kohno and 18050024 to Y. Kimata) and for 21st Century Center of Excellence Research from the Ministry of Education, Culture, Sports, Science and Technology and by a Grant-in-Aid from the Japanese Society for the Promotion of Science (KAKENHI 18570179 to Y. Kimata).
Submitted: 26 April 2007
Accepted: 11 September 2007
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Bertolotti, A., Y. Zhang, L.M. Hendershot, H.P. Harding, and D. Ron. 2000. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat. Cell Biol. 2:326–332.[CrossRef][Medline]
Christianson, T.W., R.S. Sikorski, M. Dante, J.H. Shero, and P. Hieter. 1992. Multifunctional yeast high-copy-number shuttle vectors. Gene. 110:119–122.[CrossRef][Medline]
Cox, J.S., and P. Walter. 1996. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell. 87:391–404.[CrossRef][Medline]
Cox, J.S., C.E. Shamu, and P. Walter. 1993. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 73:1197–1206.[CrossRef][Medline]
Craven, R.A., M. Egerton, and C.J. Stirling. 1996. A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of several protein precursors. EMBO J. 15:2640–2650.[Medline]
Credle, J.J., J.S. Finer-Moore, F.R. Papa, R.M. Stroud, and P. Walter. 2005. On the mechanism of sensing unfolded protein in the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 102:18773–18784.
DuRose, J.B., A.B. Tam, and M. Niwa. 2006. Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress. Mol. Biol. Cell. 17:3095–3107.
Goffin, L., S. Vodala, C. Fraser, J. Ryan, M. Timms, S. Meusburger, B. Catimel, E.C. Nice, P.A. Silver, C.Y. Xiao, et al. 2006. The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins. Mol. Biol. Cell. 17:5309–5323.
Harding, H.P., Y. Zhang, and D. Ron. 1999. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 397:271–274.[CrossRef][Medline]
Haze, K., H. Yoshida, H. Yanagi, T. Yura, and K. Mori. 1999. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol. Biol. Cell. 10:3787–3799.
Higashio, H., Y. Kimata, T. Kiriyama, A. Hirata, and K. Kohno. 2000. Sfb2p, a yeast protein related to Sec24p, can function as a constituent of COPII coats required for vesicle budding from the endoplasmic reticulum. J. Biol. Chem. 275:17900–17908.
Kimata, Y., Y.I. Kimata, Y. Shimizu, H. Abe, I.C. Farcasanu, M. Takeuchi, M.D. Rose, and K. Kohno. 2003. Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. Mol. Biol. Cell. 14:2559–2569.
Kimata, Y., D. Oikawa, Y. Shimizu, Y. Ishiwata-Kimata, and K. Kohno. 2004. A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1. J. Cell Biol. 167:445–456.
Kimata, Y., Y. Ishiwata-Kimata, S. Yamada, and K. Kohno. 2006. Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins. Genes Cells. 11:59–69.
Kohno, K. 2007. How transmembrane proteins sense endoplasmic reticulum stress. Antioxid. Redox Signal. In press.
Kohno, K., K. Normington, J. Sambrook, M.J. Gething, and K. Mori. 1993. The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol. Cell. Biol. 13:877–890.
Lipson, K.L., S.G. Fonseca, S. Ishigaki, L.X. Nguyen, E. Foss, R. Bortell, A.A. Rossini, and F. Urano. 2006. Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1. Cell Metab. 4:245–254.[CrossRef][Medline]
Liu, C.Y., M. Schroder, and R.J. Kaufman. 2000. Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum. J. Biol. Chem. 275:24881–24885.
Liu, C.Y., H.N. Wong, J.A. Schauerte, and R.J. Kaufman. 2002. The protein kinase/endoribonuclease IRE1alpha that signals the unfolded protein response has a luminal N-terminal ligand-independent dimerization domain. J. Biol. Chem. 277:18346–18356.
Ma, K., K.M. Vattem, and R.C. Wek. 2002. Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J. Biol. Chem. 277:18728–18735.
Mori, K., A. Sant, K. Kohno, K. Normington, M.J. Gething, and J.F. Sambrook. 1992. A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. EMBO J. 11:2583–2593.[Medline]
Mori, K., W. Ma, M.J. Gething, and J. Sambrook. 1993. A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 74:743–756.[CrossRef][Medline]
Nadanaka, S., T. Okada, H. Yoshida, and K. Mori. 2007. Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress. Mol. Cell. Biol. 27:1027–1043.
Oikawa, D., Y. Kimata, M. Takeuchi, and K. Kohno. 2005. An essential dimer- forming subregion of the endoplasmic reticulum stress sensor Ire1. Biochem. J. 391:135–142.[CrossRef][Medline]
Oikawa, D., Y. Kimata, and K. Kohno. 2007. Self-association and BiP dissociation are not sufficient for activation of the ER stress sensor Ire1. J. Cell Sci. 120:1681–1688.
Oka, M., M. Nakai, T. Endo, C.R. Lim, Y. Kimata, and K. Kohno. 1998. Loss of Hsp70-Hsp40 chaperone activity causes abnormal nuclear distribution and aberrant microtubule formation in M-phase of Saccharomyces cerevisiae. J. Biol. Chem. 273:29727–29737.
Okamura, K., Y. Kimata, H. Higashio, A. Tsuru, and K. Kohno. 2000. Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast. Biochem. Biophys. Res. Commun. 279:445–450.[CrossRef][Medline]
Papa, F.R., C. Zhang, K. Shokat, and P. Walter. 2003. Bypassing a kinase activity with an ATP-competitive drug. Science. 302:1533–1537.
Sato, K., M. Sato, and A. Nakano. 2003. Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes. Mol. Biol. Cell. 14:3605–3616.
Shamu, C.E., and P. Walter. 1996. Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. EMBO J. 15:3028–3039.[Medline]
Shen, J., X. Chen, L. Hendershot, and R. Prywes. 2002. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev. Cell. 3:99–111.[CrossRef][Medline]
Sidrauski, C., and P. Walter. 1997. The transmembrane kinase Ire1p is a site- specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell. 90:1031–1039.[CrossRef][Medline]
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:19–27.
Sun, G.H., A. Hirata, Y. Ohya, and Y. Anraku. 1992. Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity. J. Cell Biol. 119:1625–1639.
Travers, K.J., C.K. Patil, L. Wodicka, D.J. Lockhart, J.S. Weissman, and P. Walter. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 101:249–258.[CrossRef][Medline]
Zhou, J., C.Y. Liu, S.H. Back, R.L. Clark, D. Peisach, Z. Xu, and R.J. Kaufman. 2006. The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response. Proc. Natl. Acad. Sci. USA. 103:14343–14348.
CiteULike 
Complore 
Connotea 
Del.icio.us 
