Here, we demonstrate that Ire1 clusters when activated. Impairment of the cluster formation, either by the F247A or the W426A mutation, strongly suggests that the molecular basis of the cluster formation is high order oligomerization of the CSSR (Credle et al. 2005
). Importantly and conversely, our finding provides evidence for the high order oligomerization of Ire1, which, as detailed in the Introduction, has been unsupported by many of the previous biochemical analyses of cell lysates or solutions of recombinant Ire1 fragments. We think that the homomeric interaction via interface II is so weak that the association is undetectable in samples used in the biochemical analyses, where the concentration of Ire1 is probably much lower than its local concentration on the ER membrane. It is likely that cluster formation is not a result but a prerequisite of activation of Ire1. This is because Ire1 clustered even with the kinase mutation K702A or in hac1
Δ 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 ΔIII 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 ΔIΔV Ire1 was constitutively clustered, but extrinsic ER stress was still required for full activation of this mutant. Furthermore, the S103P and the ΔLLSK mutations are likely to abolish the requirement of Step 2 for activation of Ire1; in contrast, the ΔIII 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 ΔIΔV Ire1, either ΔI or ΔV single mutant showed normal ER-like localization. This finding indicates that the cluster formation requires both dissociation of BiP or the BiP-nonbinding mutation ΔV, 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 ΔIΔV Ire1 undergoes regulation in Step 2 and because none of the Step 2 mutants (ΔIII, S103P, or ΔLLSK) affect BiP binding and its dissociation from Ire1. Impairment of the interaction between denatured proteins and the CSSR protein carrying the ΔIII mutation strongly suggests that Step 2 is regulated by direct interaction of unfolded proteins with Ire1. Considering the constitutive cluster formation of ΔIΔV 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 ΔLLSK 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 , 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.
As a result of this work, we propose a model that is illustrated in . Importantly, ER stress provokes multiple events that separately contribute to activation of Ire1 at different steps. Cluster formation probably leads to considerably higher local concentration of the cytosolic effector domain of Ire1, which may be required for efficient cleavage of the HAC1u
mRNA. Regulation by dual steps in different manners is likely to be important for precision of response by ensuring that Ire1 is only up-regulated by ER stress. Indeed, either ethanol or high temperature inappropriately activates ΔV Ire1 (Kimata et al., 2004
Nevertheless, it is likely that not all events noted in are required to obtain partial activation of Ire1. Indeed, the constitutively clustering mutant ΔIΔV 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 IRE1α in pancreatic β 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 IRE1α, 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 IRE1α 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)