In contrast to the classical model, in which unfolded proteins accumulated in the endoplasmic reticulum trigger the unfolded-protein response (UPR), we show that membrane aberrancy also evokes this protective cellular event. This finding may explain UPR activation under various physiological conditions.

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.

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.

The unfolded protein response (UPR) is a cellular protective event against endoplasmic reticulum (ER) stress. In the yeast UPR signaling pathway, the ER-located transmembrane protein Ire1 promotes splicing of the HAC1 premRNA ( HAC1 u ) to produce the translatable transcription factor mRNA ( HAC1 i ). We generated a HAC1 i gene-bearing strain, in which the UPR pathway was constitutively activated, and compared its gene expression profile with that of a ∆ ∆ ∆ ∆ ire1 HAC1 u strain using cDNA microarray technology. Comparison of the gene expression profile was also performed between non-stressed wild-type cells and those exposed to ER stress. Genes for which the expression level was significantly changed in both of these experiments were categorized as targets of the Ire1-HAC1 signaling pathway. This analysis revealed that in addition to the previously known UPR targets, some anti-oxidative stress genes were up-regulated by the Ire1-HAC1 pathway, possibly in order to reduce reactive oxygen species produced during the cellular response to ER stress. Moreover, we categorized 15 genes as those down-regulated by the UPR, most of which seem to encode cell surface or extracellular proteins. This UPR-mediated gene repression may alleviate the load of client proteins targeted to the ER.

Cellular exposure to cadmium is known to strongly induce the unfolded protein response (UPR), which suggests that the endoplasmic reticulum (ER) is preferentially damaged by cadmium. According to recent reports, the UPR is induced both dependent on and independently of accumulation of unfolded proteins in the ER. In order to understand the toxic mechanism of cadmium, here we investigated how cadmium exposure leads to Ire1 activation, which triggers the UPR, using yeast Saccharomyces cerevisiae as a model organism. Cadmium poorly induced the UPR when Ire1 carried a mutation that impairs its ability to recognize unfolded proteins. Ire1 activation by cadmium was also attenuated by the chemical chaperone 4-phenylbutyrate. Cadmium caused sedimentation of BiP, the molecular chaperone in the ER, which suggests the ER accumulation of unfolded proteins. A green fluorescent protein-based reporter assay also indicated that cadmium damages the oxidative protein folding in the ER. We also found that an excess concentration of extracellular calcium attenuates the Ire1 activation by cadmium. Taken together, we propose that cadmium exposure leads to the UPR induction through impairment of protein folding in the ER.

In eukaryotic cells under nonstressed conditions, the endoplasmic reticulum (ER)‐located molecular chaperone BiP is associated with an ER‐membrane protein Ire1 to inhibit its self‐association. While ER stress leads Ire1 to form transiently BiP‐unbound clusters, which strongly evoke the unfolded protein response (UPR), here we propose an alternative activation status of Ire1. When yeast cells are physiologically ER‐stressed by inositol depletion for a prolonged time, the UPR is weakly activated in a sustained manner after a transient peak of activation. During persistent stress, Ire1 foci disappear, while Ire1 continues to be self‐associated. Under these conditions, Ire1 may be activated as a homo‐dimer, as it shows considerable activity even when carrying the W426A mutation, which allows Ire1 to form homo‐dimers but not clusters. Unlike the Ire1 clusters, the nonclustered active form seems to be associated with BiP. An Ire1 mutant not carrying the BiP‐association site continued to form clusters and to be activated strongly even after long‐term stress. Similar observations were obtained when cells were ER‐stressed by dithiothreitol. We thus propose that upon persistent ER stress, Ire1 is weakly and continuously activated in a nonclustered form through its (re)association with BiP, which disperses the Ire1 clusters.

Accumulation of unfolded secretory proteins in the endoplasmic reticulum (ER), namely ER stress, is hazardous to eukaryotic cells and promotes the unfolded protein response (UPR). Ire1 is an ER-located transmembrane protein that senses ER stress and triggers the UPR. According to previous in vitro experiments, 4-phenylbutyrate (4-PBA) works as a chemical molecular chaperone. Since 4-PBA attenuates the UPR in mammalian tissue cultures, this chemical may have clinical potential for restoring ER-stressing conditions. In this study, we investigated 4-PBA's mode of action using the yeast Saccharomyces cerevisiae as a model organism. Although 4-PBA blocked a dithiothreitol (DTT)-induced UPR, it did not appear to restore impairment of ER protein folding that was caused by DTT. Moreover, even under non-stress conditions, 4-PBA attenuated UPR that was induced by an Ire1 mutant that exhibits a substantial activity without sensing ER accumulation of unfolded proteins. We also found that 4-PBA drastically promotes the degradation of Ire1. These observations indicate that at least in the case of yeast cells, 4-PBA suppresses the UPR not through restoration of the ER function to correctly fold proteins. Instead, the accelerated degradation of Ire1 possibly explains the reason why the UPR is attenuated by 4-PBA.

ABSTRACT. Endoplasmic reticulum (ER) stress causes the ER-resident transmembrane protein Ire1 to selfassociate, leading to the formation of large oligomeric clusters. In yeast cells, this induces strong unfolded protein response (UPR) through splicing of HAC1 mRNA. Here, we demonstrate that highly ER-stressed yeast cells exhibited poor Ire1 clustering in the presence of the actin-disrupting agent latrunculin-A. Under these conditions, Ire1 may form smaller oligomers because latrunculin-A only partially diminished the Ire1-mediated splicing of HAC1 mRNA. Ire1 cluster formation was also impaired by deletion of the type-II myosin gene MYO1 or SAC6, which encodes the actin-bundling protein fimbrin. Finally, we demonstrated that Ire1 clusters are predominantly located on or near actin filaments. Therefore, we propose that actin filaments play an important role in ER stressinduced clustering of Ire1.