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IRE1: ER stress sensor and cell fateexecutorYani Chen and Federica Brandizzi

MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, 612 Wilson Rd, Room 122,

East Lansing, MI 48824, USA

Review

Cells operate a signaling network termed the unfoldedprotein response (UPR) to monitor protein-folding capac-ity in the endoplasmic reticulum (ER). Inositol-requiringenzyme 1 (IRE1) is an ER transmembrane sensor thatactivates the UPR to maintain the ER and cellular function.Although mammalian IRE1 promotes cell survival, it caninitiate apoptosis via decay of antiapoptotic miRNAs.Convergent and divergent IRE1 characteristics betweenplants and animals underscore its significance in cellularhomeostasis. This review provides an updated scenarioof the IRE1 signaling model, discusses emerging IRE1sensing mechanisms, compares IRE1 features amongspecies, and outlines exciting future directions in UPRresearch.

ER stress and the UPRThe membrane trafficking system maintains the operationof approximately one-third of the eukaryotic proteome.Most secretory proteins first enter the ER for foldingand assembly. To maintain the fidelity of ER functions,cells coordinate a protein quality-control system with asignaling network, the UPR [1–6]. The UPR is triggered byER transmembrane sensors on ER stress, a cellular condi-tion referring to the accumulation of unfolded proteinsin the ER (Figure 1) [7,8]. The adaptive response occurringat the initial phase of the UPR aims to rebalance protein-folding homeostasis [2,3,9–12]. If cells fail to recover fromER stress, the UPR represses the adaptive response andtriggers apoptosis [1,3,4,7,13,14]. IRE1 is the only identi-fied ER stress sensor in yeast and is essential for theUPR in animals and plants [15–18] (Figure 1). As an ERtransmembrane protein, IRE1 monitors ER homeostasisthrough an ER luminal stress-sensing domain and triggersthe UPR through a cytoplasmic kinase domain and anRNase domain [15,16]. On ER stress, IRE1 RNase isactivated through conformational change, autophosphor-ylation, and higher-order oligomerization [19–21]. Mam-malian IRE1 initiates diverse downstream signaling of theUPR either through unconventional splicing of the tran-scription factor Xbp-1 or through post-transcriptional mod-ifications via Regulated IRE1-Dependent Decay (RIDD) ofmultiple substrates [15,16,22–25]. In addition, PERK andATF6 function as two distinct mammalian ER-stress sen-

0962-8924/$ – see front matter

� 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.06.005

Corresponding author: Brandizzi, F. (fb@msu.edu).Keywords: unfolded protein response; ER stress; IRE1; cell fate;protein quality control; membrane trafficking system.

sors to cope with complex UPR scenarios [7,26] (Figure 1).Similar to IRE1, PERK, and ATF6 are ER transmembraneproteins that contain an ER luminal stress-sensing domainand a cytoplasmic enzymatic domain. To prevent a furtherincrease in protein-folding demand in the ER, PERK tran-siently inhibits general protein translation through phos-phorylation of eukaryotic initiation factor 2 alpha (eIF2a).Phosphorylated elF2a can also selectively activate trans-lation of mRNAs including the ATF4 transcription factor toregulate UPR target genes [27]. ER stress triggers reloca-tion of ATF6 from the ER to the Golgi, where it undergoesproteolytic cleavage. The cleaved transcription factordomain of ATF6 enters the nucleus for UPR regulation[28–30] (Figure 1).

The main molecular mechanisms underlying IRE1 un-conventional splicing are conserved in eukaryotes. Inbudding yeast, mammals, and plants, there is only onetranscription factor identified as a splicing target ofIRE1 (Figure 1). The stem–loop structure and cleavagesite of the IRE1-splicing substrate are conserved amongspecies. By contrast, RIDD appears more divergent ineukaryotes. Among yeast, RIDD operates in the fissionyeast Schizosaccharomyces pombe but not in the buddingyeast Saccharomyces cerevisiae [31]. Intriguingly, RIDD-mediated decrease in protein-folding demand is the onlyidentified mechanism of UPR in fission yeast [31]. Al-though plant RIDD may potentially degrade a significantportion of mRNAs encoding secretory proteins [32], it isundetermined whether plant RIDD processes various sub-strates to direct UPR outputs like mammalian RIDD.Unlike the mammalian UPR, plant PERK orthologs re-main to be identified; however, two functional homologs ofATF6, bZIP28, and bZIP17, exist in plants [33] (Figure 1).Moreover, a component of the G protein complex, AGB1,is essential for the plant UPR [17] and an alternativeG-protein-coupled receptor is involved in a non-canonicalUPR in Caenorhabditis elegans [34]. Due to the largenumber of members of the mammalian G protein complex,its roles in the classical UPR might be more challenging toreveal. Although the IRE1 and ATF6 arms are partiallyconserved between plants and animals, it will be interest-ing to establish the degree of UPR diversification betweenthe two kingdoms.

This review presents the latest advances in and view-points on IRE1-dependent UPR research. We focus on therecent groundbreaking discoveries that define IRE1 as amaster regulator in cell fate determination under ERstress. IRE1 was long considered a positive regulator of

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P

Selec�vetransla�on

Transla�ona�enua�on

AGB1

Decrease in proteinloading into ER

Proteolysis

P P

mRNAdecay

ER lumen

Splicing

ATF4

elF2α PelF2α

Cytoplasm

PERK1 IRE1

Unspliced bZIP

Spliced bZIP

bZIP

Golgilumen

UPR target genes

Nucleus

ATF6bZIP17, bZIP28

CHOP

Apoptosis

JNKP

Apoptosis

PP

Xbp-1bZIP60Hac1

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Figure 1. Overview of unfolded protein response (UPR) arms in eukaryotes. The inositol-requiring enzyme 1 (IRE1) arm is conserved in eukaryotes. IRE1 unconventionally

splices the bZIP transcription factors Xbp-1 and bZIP60 and Hac1 mRNA in mammals, plants, and yeast, respectively. The spliced bZIP transcription factor enters into the

nucleus to regulate UPR target genes. In addition, two distinct arms mediated by PERK and ATF6 regulate the mammalian UPR. ATF6 is an endoplasmic reticulum (ER)

transmembrane transcription factor. ER stress triggers the relocation of ATF6 from the ER to the Golgi apparatus, where it undergoes proteolytic cleavage. Subsequently,

the transcription factor domain of ATF6 enters into the nucleus to modulate transcription of UPR target genes. Two functional homologs of ATF6, bZIP17, and bZIP28, exist

in plants. PERK, an ER transmembrane protein kinase, is identified only in animals. On ER stress, PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2a), which

leads to transient inhibition of general protein translation and selective translation of the transcription factor ATF4. Under irremediable ER stress, PERK–elF2a–ATF4–CHOP

and IRE1–JNK initiate apoptosis in mammals. Moreover, the beta subunit of the heterotrimeric G protein complex AGB1 is essential for the plant UPR. Although the

G protein complex is conserved in eukaryotes, its significance in the UPR is unclear in other eukaryotic organisms. Blue, eukaryotes; black, mammals; green, plants;

red, yeast.

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cell survival. Thus, repression of IRE1 was believed topotentiate apoptosis. The recent identification of novelIRE1 regulatory events reveals that IRE1 signaling ispersistent during ER stress. Namely, IRE1 can no longerbe considered simply as a driving force for cell survival, butrather as an administrator/executor of cell fate determina-tion under ER stress. Through presentation of the recentevidence establishing that IRE1 triggers diverse signaling,

2

we delineate current IRE1-signaling models. It has alsobecome clear that IRE1 monitors cellular homeostasisbeyond protein-folding status in the ER; therefore, thefunctional relevance of the UPR within physiological pro-cesses is discussed. Finally, we compare convergent anddivergent features of IRE1 between plants and mammalsto provide an integrated view of IRE1 in multicellulareukaryotes.

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IRE1 signaling in cell fate determinationLife versus death determination is constantly scrutinizedand tightly controlled. The prevalence of malfunctioningcells due to irremediable ER stress contributes to signifi-cant diseases, including cancer and diabetes. Conversely,overcommitment to cell death may result in organ damageor cell-degenerative diseases [35–39]. To reach optimalfitness under ER stress, cell fates are determined throughtight coordination of adaptive and apoptotic responses[37,40,41]. In mammals, PERK–eIF2a–ATF4 regulatesthe transcription factor CHOP to activate ER stress-triggered apoptosis. In parallel, IRE1 controls cell fatedetermination through the mitogen protein kinase JNKunder ER stress [3,7,8,42] (Figure 1). By contrast, althoughER stress can play a role in programmed cell death inplants [43], very little is known about ER stress-inducedcell death in plants [17,32,44]. Furthermore, lack of se-quence homologs of most mammalian apoptosis regulatorsin plants hints that divergent mechanisms of ER stress-induced cell death exist among organisms.

Revised model of the IRE1a signaling network

in mammals

The mammalian genome encodes two isoforms of IRE1,IRE1a and IRE1b. IRE1a is expressed ubiquitously andIRE1a knockout mice exhibit embryonic lethality. By con-trast, IRE1b expression is restricted and IRE1b knockoutmice are viable [45,46]; therefore, most mammalian UPRresearch is conducted on IRE1a. IRE1a was identified as apositive regulator of cell survival. It was believed thatIRE1a signaling was terminated during irremediable ERstress to enable apoptosis [1,2,7,15,47,48]. Nevertheless,recent studies have challenged this concept by showingthat IRE1a persistently adjusts protein-folding capacity,actively directs UPR signaling, and executes cell fatedetermination [49,50] (Figure 2). IRE1a employs splicingand RIDD to direct cell fate throughout ER stress. DespiteXbp-1 being the only identified IRE1a splicing target,numerous types of RNA are proven RIDD substrates[22,49,50]. Although the significance of RIDD targets isnot completely understood, some RIDD events are criticalfor IRE1a-dependent cell fate determination. During theadaptive response, IRE1a conducts RIDD on mRNAsencoding ER-translocating proteins to prevent furtherincreases in protein-folding demand in the ER [50]. Toaugment protein-folding ability, IRE1a splices the tran-scription factor Xbp-1 mRNA to induce the transcription ofER quality-control components. If attempts to restore ERhomeostasis fail, IRE1a ceases to splice Xbp-1 mRNA.Alternatively, IRE1a represses adaptive responses andactivates apoptosis through RIDD [49,50]. During thetransition phase, occurring between the adaptive and apo-ptotic response, RIDD increases ER stress intensitythrough degradation of selective UPR target genes includ-ing the ER protein chaperone BiP [50]. Once the ER stressintensity reaches its threshold, RIDD initiates apoptosisthrough repression of antiapoptotic pre-miRNAs [49]. Cas-pase-2 (CASP2) is a proapoptotic protease essential for theexecution of apoptosis [51]. Upregulation of CASP2 is anindicator of apoptotic initiation. Through decay of anti-Casp2 pre-miRNAs, IRE1a activates apoptosis through

upregulation of Casp2 (Figure 2) [49]. A close associationof IRE1a activity and cell fate determination has beenproposed for years [1,2,7,15,47,48]. These findings providedirect evidence that IRE1a is a molecular switch andapoptosis executioner during ER stress [49]. It was previ-ously proposed that attenuation of IRE1 activity allowscells to initiate apoptosis [1,2,7,15,47,48]. The identifica-tion of the IRE1a–Casp2 pathway elaborates an intriguingIRE1a signaling model: IRE1a–Xbp-1 is active in theadaptive phase and attenuated in the apoptotic phase.In parallel, activation of IRE1a–Casp2 events initiates celldeath in the apoptotic phase (Figure 3).

Is mammalian IRE1a the only major trigger of ER

stress-induced apoptosis?

IRE1a is necessary and sufficient to trigger apoptosis,whereas PERK and ATF6 are dispensable in apoptosisactivation [49]. Nonetheless, it cannot be excluded thatdistinct ER-stress sensors may serve as major executionersof cell death in a context-specific manner. Using chemicalgenetic tools, the regulatory roles of the phosphor-transferand RNase activity of IRE1a in the UPR can be examinedseparately. The phosphor-transfer function is dispensablefor Xbp-1 mRNA splicing and the upregulation of CASP2expression; however, it is required for the subsequentCASP2 cleavage and apoptosis activation, indicating thatthe IRE1a phosphor-transfer function is essential for cell-fate switching during ER stress [49,50]. Notably, the phos-phor-transfer function is mostly studied through an in vitroconditional IRE1a induction that mimics ER stress.Although this experimental system is valuable to distin-guish the phosphor-transfer and RNase functions ofIRE1a, it is important to note that ATF6 and PERK arenot activated through ER stress. A potentially compro-mised crosstalk among the UPR arms raises a possibilitythat the IRE1a induction system might not completelyresemble a genuinely biological scenario of ER stress.Hence, careful data interpretation from the conditionalinduction system and integration of in vivo analyses arenecessary to determine whether IRE1a is the mastertrigger in ER stress-induced apoptosis.

The substrate specificity of mammalian IRE1a

Although the four identified IRE1a-cleaved miRNAs(miR-17, miR-34a, miR-96, and miR-125b) repress the com-mon substrate Casp2, TXNIP is another target of miR-17[52]. TXNIP is involved in b-cell death and was selected topotentially regulate ER stress-induced apoptosis based onits rapidly elevated expression under severe ER stress.Similar to the IRE1a mutation, TXNIP mutation leadsto compromised apoptosis activation, indicating that TXNIPis essential for ER stress-induced apoptosis [52,53]. Where-as PERK-eIF2a activates TXNIP transcription, IRE1a

increases TXNIP expression by degradation of miR-17.Accordingly, it is conceivable that each of the four IRE1a-cleaved miRNAs might have specific substrates such asTXNIP. Based on this scenario, IRE1a might differentiallydegrade its individual target miRNA for fine-tuning of theUPR. Another interesting feature of mammalian RIDD isthat distinct substrates have a degree of sequence similaritywithin the cleavage site, whereas the flanking sequences of

3

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Nascent pep�de

PP P

Unfoldedprotein

Folded protein

BiP

IRE1α

Ribosome

Translocon

UPR genes

Cell survivalmRNA

mRNAdecay

Nucleus

Xbp-1uXbp-1s

ER lumen

ER stress

Splicing

Cytochrome crelease

Cell death

XBP-1

XBP-1

Cytoplasm

Mitochondrion

Cell death

Output signaling

mRNA encodingER-transloca�ngproteins

IRE1α substrates

An�-Casp2 miRNA Derepression of cell death

Three types of mRNA decay events

Decrease in protein-foldingdemand

1

3

mRNA of UPRtarget genes

Increase in ER stressintensity

2

BiP

Figure 2. Inositol-requiring enzyme 1 alpha (IRE1a) regulatory mechanisms during endoplasmic reticulum (ER) stress. Mammalian IRE1a is repressed through a physical

interaction with BiP when the demand for and capacity of protein folding is balanced in the ER. Dissociation of IRE1a from BiP due to an elevated level of unfolded protein in

the ER leads to activation of IRE1a. IRE1a-activating processes include its autophosphorylation, conformational change, and higher-order assembly. IRE1a directs cell fate

decisions through unconventional splicing and Regulated IRE1-Dependent Decay (RIDD). To prevent increasing demand for ER protein folding, IRE1a conducts RIDD to

degrade the transcripts of ER-translocating proteins. In parallel, IRE1a unconventionally splices transcripts of the Xbp-1 transcription factor. Spliced XBP-1 enters the

nucleus to transcriptionally reprogram unfolded protein response (UPR) target genes, including ER chaperones. Under irremediable ER stress, IRE1a ceases to splice Xbp-1

mRNA. Instead, IRE1a performs RIDD on selective UPR target genes including BiP to enhance the intensity of the ER stress. Once the ER stress intensity reaches its

threshold, IRE1a represses anti-Casp2 miRNA (miR-17, miR-34a, miR-96, and miR-125b) through RIDD. IRE1a-mediated degradation of anti-Casp2 miRNAs leads to

activation of the apoptotic initiator Casp2 and subsequently triggers mitochondrion-dependent apoptosis.

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4

IRE1 α-Casp2

IRE1α-Xbp-1

PERK

Adap�ve response Transi�on Apopto�c response

Dura�on of ER stress

Ac�v

a�on

leve

l of s

igna

ling

ER stress intensity

Decrease in protein-folding demand

Ac�va�on ofcell deathIncrease in protein-folding capacity

Decrease inprotein-folding

capacity

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Figure 3. Updated model of inositol-requiring enzyme 1 alpha (IRE1a) and PERK signaling in cell fate determination during endoplasmic reticulum (ER) stress. Unfolded

protein response (UPR) signaling aimed at cell survival is considered an adaptive response during ER stress. Under irremediable ER stress, the UPR represses the adaptive

response and triggers an apoptotic response. IRE1a and PERK are two ER stress sensors that decrease ER protein-folding demand through mRNA decay and translational

inhibition, respectively. Both PERK and IRE1a signaling appear to persist throughout ER stress. IRE1a differentially triggers diverse UPRs according to need. In the adaptive

phase, to increase protein-folding capcity, IRE1a-mediated Xbp-1 mRNA splicing is activated for transcriptional regulation of UPR target genes. In a transition phase

between the adaptive and apoptotic responses, the signaling mediated by IRE1a–Xbp-1 is attenuated. In parallel, IRE1a increases the intensity of the ER stress through

mRNA decay of selective UPR target genes, including ER chaperones. During the apoptotic phase, IRE1a–Casp2 signaling is activated to initate cell death.

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the cleavage sites are relatively divergent [49,54]. Thissuggests that the cleavage mechanisms are likely to beconserved, whereas the flanking sequence determines thespecificity of substrate recognition. This scenario wouldsupport the hypothesis that IRE1a adjusts its RNase sub-strate specificity to activate diverse UPRs. The flexibility ofIRE1a to target different substrates might rely on combina-tions of phosphorylation status, conformational changes,and physical associations with IRE1a regulators. Becausealterations of IRE1a substrate specificity lead to oppositecell fates [50], further understanding of IRE1a substratepreferences will reveal how IRE1a coordinates cellularhomeostasis to determine cell fate under ER stress.Currently, target switching of RIDD has been reported onlyin animals. Therefore, to gain a deeper understanding ofthe evolution of the UPR in eukaryotes, further studies areneeded to determine whether similar mechanisms exist inyeast and plants.

Plant IRE1 in the ER stress response and cell fate

determination

Despite the conservation of IRE1 among eukaryotes, di-vergent IRE1-dependent regulatory events have also beenobserved between plants and mammals. These evolution-arily divergent mechanisms are likely to be the reason forthe different ER stress and cell fate phenotypes observedbetween plants and mammals. Unlike mammalian IRE1isoforms, the two Arabidopsis IRE1 isoforms are expressedubiquitously with a limited tissue-specific expression pat-tern [55,56]. There is no significant defect of the UPR insingle mutants of Arabidopsis IRE1A or IRE1B, whereasArabidopsis ire1 double mutants display compromised ERstress tolerance and a UPR activation phenotype [17,18].These observations indicate that the two Arabidopsis IRE1homolog share partially overlapping function during theUPR. Evidence for established, dominant, or specific rolesof individual Arabidopsis IRE1 isoforms during the UPRand cell fate regulation need to be further elucidated.

Notably, it is experimentally undetermined whether viableArabidopsis ire1b are knockouts or partial loss-of-functionmutants. Failure to recover a homozygous plant of putativeIRE1B knockout hints that Arabidopsis IRE1B might bean essential gene similar to mammalian IRE1a [57]. In-terestingly, although mammalian IRE1a is essential forthe UPR in goblet cells, in other cell types there is nodetectable defect in UPR target gene induction in a mam-malian ire1 double mutant, probably due to partially over-lapping function with ATF6 and PERK [46,58]. Bycontrast, although two functional homologs of ATF6,bZIP28 and bZIP17, exist in Arabidopsis [33] (Figure 1),Arabidopsis ire1 double mutants exhibit a dramatic reduc-tion of UPR target gene activation [17,18]. These dataindicate that the UPR is partially diversified betweenmammals and plants. Nonetheless, similar IRE1 featuresare also observed between plants and mammals. For in-stance, ire1 and xbp1-1 mutants display differential phe-notypes despite both being essential genes. Likewise,mutants of the Arabidopsis IRE1 splicing target bZIP60show ER stress tolerance comparable with wild type plantsas opposed to Arabidopsis ire1 double mutants [17], sup-porting the hypothesis that the function of Arabidopsis IRE1is not restricted to unconventional splicing like mammalianIRE1.

Interestingly, mutations of IRE1 in plants and mam-mals lead to opposite effects in ER stress-induced celldeath phenotypes [17,18,32]. Ire1a�/� mouse embryonicfibroblasts (MEFs) exhibit a greater survival rate thanIre1a+/+ MEFs under ER stress, supporting the suggestionthat mammalian IRE1 is an apoptosis executioner. Bycontrast, Arabidopsis ire1 double mutants display compro-mised ER stress tolerance, instead of a greater survivalrate [17,18]. Consistently, DNA fragmentation and ionleakage are enhanced in the Arabidopsis ire1 double mu-tant during ER stress [32], suggesting that plant IRE1might not function as an apoptosis executioner likeits mammalian counterpart. Nevertheless, it cannot be

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excluded that the differences are related to dissimilarexperimental settings; mammalian UPR research is most-ly conducted in cell culture, whereas intact organisms areused in plant UPR studies. Moreover, except for potentialroles in protein-loading reduction under ER stress [32], thebiological significance of plant RIDD in cell fate determi-nation is unknown. Further experimental validation willreveal whether plant RIDD could process multiple sub-strates to control cell fate decisions, similar to that seen inmammals.

Shared components of the UPRosome and apoptosisIRE1a activation is tightly controlled by its interactingprotein complex, termed the UPRosome [15]. Most UPRo-some components are involved in apoptosis, supporting thesuggestion that intense crosstalk exists between IRE1a

activity and apoptosis activation (Table 1). Specifically,although the UPRosome comprises multiple components,loss-of-function mutation of a single component, such asPARP16, Bi-1, Aip-1, PTP-1B, NMHCIIB, Jab1, Nck1, orAsk1, is sufficient to alter IRE1a splicing activity or apo-ptosis activation [59–66] (Table 1). Moreover, IRE1a-interactor mutants displaying either elevated or declinedIRE1a splicing activity can show enhanced apoptosis,indicating that a precise level of activation of IRE1a splic-ing is important for cell survival [59–66]. This furthersuggests that IRE1a activation is controlled by a signalingnetwork that maintains a delicate equilibrium of adaptiveand apoptotic responses. A subtle imbalance of the equi-librium could disturb cellular homeostasis and thus altercell fate determination [59–76]. Furthermore, the observa-tion that a single mutation of the UPRosome leads tosignificant defects in IRE1a signaling hints that IRE1a

is differentially regulated in a context-specific manner(Table 1). Because UPRosome analyses are conductedunder various conditions, systematic and comparable anal-yses of UPRosome members will connect each hub and thusgive a clearer view of the IRE1a signaling network.

Table 1. Interacting proteins of IRE1a

IRE1a interactors Function of IRE1a interactors Observ

PARP16 Poly ADP-ribose polymerase Decrea

Bi-1 Anti-apoptotic protein Increas

Aip-1 Transducer of apoptotic signaling Decrea

Ptp-1b Protein tyrosine phosphatase 1B Decrea

NMHCIIB Myosin cytoskeleton Decrea

Bax/Bak Pro-apoptotic protein Decrea

Bim/Puma Pro-apoptotic protein Decrea

Jab1 Apoptosis-related protein Decrea

Nck1 SH2/SH3 domain containing adaptor Decrea

Ask1 Apoptosis signal-regulated kinase Decrea

IRE1a interactors Function of IRE1a interactors Observ

Traf2 Tumor necrosis factor Decrea

TRAF2

Jik JNK inhibitory kinase Increas

Hsp90 Heat shock protein Decrea

Usp14 Ubiquitin specific peptidase Increas

Usp14

SYVN1 E3 ubiquitin ligase/anti-apoptotic factor Increas

IRE1 a

Hsp72 Heat shock protein Increas

Rack1 Scaffold protein for activated protein kinase Decrea

6

IRE1 sensing mechanismsER stress-sensing mechanisms are intensively studied inyeast and animals [77]. The ER stress sensors are silentthrough physical association with BiP, the most abundantER-resident chaperone. Dissociation with BiP or interac-tion with unfolded proteins is the major trigger of IRE1activation. Yeast IRE1 is activated through associationwith unfolded proteins rather than disassociation withBiP [78]; however, the physical interaction of BiP is afine-tuning mechanism to ensure that yeast IRE1 is ap-propriately activated [79]. Unlike yeast IRE1, the activa-tion mechanisms of mammalian IRE1a rely on dissociationwith BiP as opposed to a direct interaction with unfoldedproteins [80]. The differences in activation mechanismsbetween yeast and mammalian IRE1a can be partiallyexplained by the dissimilarity in protein structure withinthe sensor domain [15]. Surprisingly, a recent studyrevealed that mammalian IRE1b tends to interact withunfolded proteins like yeast IRE1 and is unable to associ-ate with BiP [81]. Accordingly, it is possible that, like yeastIRE1, binding of unfolded proteins is the primary trigger ofmammalian IRE1b activation. Despite intense studies inmammals and yeast, plant IRE1 sensing mechanisms arecompletely undefined. Further structural and functionalanalyses of plant IRE1 will be instrumental in revealingER stress-sensing mechanisms in plants.

Cell type-specific sensing mechanisms: the role of

mammalian IRE1b

How cellular homeostasis is maintained in a cell type-specific manner is a fundamental question of cell biology.It has been recently shown that IRE1b is essential for theUPR specifically in goblet cells [46]. In goblet cells, IRE1b

is dispensable for Xbp-1 splicing and BiP induction. In-stead, IRE1b mutation leads to enhanced ER stress inten-sity evidenced by higher levels of Xbp-1 splicing and BiPinduction. Moreover, IRE1b�/� mice display a distendedER phenotype, potentially due to overaccumulation of

ed phenotype of loss-of-function mutations Refs

sed Xbp-1 splicing/increased cell death [59]

ed Xbp-1 splicing/increased cell death [60]

sed Xbp-1 splicing/decreased cell death [61]

sed Xbp-1 splicing/decreased cell death [62]

sed Xbp-1 splicing/compromised IRE1a foci formation [63]

sed Xbp-1 splicing/impaired IRE1a oligomerization [67]

sed Xbp-1 splicing and UPR target genes activation [76]

sed Xbp-1 splicing and UPR target genes activation [64]

sed cell death [65]

sed cell death/altered JNK activation [66]

ed phenotype of induction, overexpression, or inhibition Refs

sed JNK activation by expression of dominant-negative [72]

ed JNK activation by overexpression of JIK [68]

sed IRE1a protein stability by treatment of HSP90 inhibitors [71]

e activity of ERAD by small interfering RNA silencing of [74]

ed IRE1 ubiquitination and degradation by coexpression of

nd SYVN1

[73]

ed Xbp-1 splicing/decreased cell death by induction of HSP72 [70]

sed IRE1a phosphorylation by overexpression of RACK1 [75]

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Mucin2 (MUC2), the most prominent protein secreted fromgoblet cells. This suggests that IRE1b controls MUC2expression in goblet cells. Thus, IRE1b mutation leadsto MUC2 overload in the ER and in turns triggers ERstress [46]. RIDD was proposed to be the mechanismunderlying IRE1b regulation of MUC2 levels in gobletcells [46]. The cell type-specific target of IRE1b providesa molecular explanation of how the UPR maintains adynamic and specific secretory ability in multicellularorganisms. Consistent with the notion that unfolded pro-teins trigger IRE1b activation, IRE1b might interact phys-ically with specific types of unfolded protein. In the case ofgoblet cells, IRE1b might specifically monitor the MUC2level in the ER and adjusts its loading into the ER throughRIDD. Based on this scenario, mammalian ER stresssensors might distinguish the type of unfolded proteinaccumulated in the ER and trigger differential UPR signal-ing. More specifically, if the unfolded proteins are dispens-able for cell survival, ER stress sensors could repress theexpression of unfolded proteins through RNA decay ortranslational repression. Conversely, if unfolded proteinsare essential for cellular function, the UPR might preferen-tially augment the expression of chaperones to recover theproduction of unfolded proteins. Although ER stress dura-tion and intensity are considered major factors in the apo-ptosis threshold, the type of misfolded protein might be alsocritical for the determination of UPR signaling outputs.

Sensing mechanisms beyond protein-folding

homeostasis

Emerging evidence shows that IRE1 monitors cellularhomeostasis beyond sensing unfolded protein accumulation.For instance, CRY1/CRY2-mediated circadian rhythmregulates IRE1a activity in the liver [82], suggesting thatIRE1a coordinates ER function to cope with circadian-relat-ed physiological processes. These observations provide alink between the IRE1a-dependent UPR, circadian regula-tion, and liver metabolic processes. More importantly,because circadian rhythm has a substantial influence onUPR activation, time-course studies of the UPR will requirediligent experimental design and appropriate controlsto avoid biases. Recently, lipid homeostasis was proven toimpact UPR activation through an unconventional sensingmechanism, because the unfolded protein-sensing domainof IRE1a and PERK is dispensable for lipid-dependent UPRactivation [83]. Together, these observations support thesuggestion that the UPR perceives physiological andcellular signaling beyond ER protein-folding homeostasis.Although it is unclear whether plant IRE1 senses signalingbeyond protein-folding capacity, an Arabidopsis ire1double mutant displaying a root-specific phenotype underunstressed conditions hints that plant IRE1 also integratesphysiological signals to maintain specific secretory activity[17]; however, this hypothesis awaits experimentalvalidation.

Concluding remarksSignificant progress in defining IRE1 mechanisms hasbeen achieved. We now know that IRE1 activities arecoordinated at a systemic level to cope with dynamicsecretion activity. Although in vitro experimental systems

and conditional IRE1 induction approaches have madegroundbreaking discoveries in basic UPR knowledge[49,50,84–87], we remain far from a comprehensive under-standing of the UPR in intact organisms. The lethality ofthe mammalian IRE1a mutant represents a challenge togaining insights into IRE1 function in vivo. By contrast,the viability of plant IRE1 mutants enables in vivo anal-yses to reveal its roles in organ growth, pathogen defense,and abiotic stress responses [17,33,88,89]. Moreover, withthe ease of building high-order plant mutants, in vivophenotypic analyses show that Arabidopsis IRE1 and aconserved component of the G protein complex display asynergistic effect in both plant UPR activation and growthregulation. This study underscores the building of UPRnetworks in intact organisms using plants as a modelsystem [17]. With more systematic and quantitative stud-ies of the UPR in vivo, there are significant findings aheadthat will decipher dynamic UPR maps close to a genuinelyphysiological scenario.

AcknowledgmentsThe authors thank Dr Danielle Loughlin for helpful comments andsuggestions. They apologize to those authors whose work could not becited owing to space constraints. This work was supported by grants fromthe National Institutes of Health (R01 GM101038-01), Chemical Sciences,Geosciences, and Biosciences Division, Office of Basic Energy Sciences,Office of Science, U.S. DOE (DE-FG02-91ER20021), NASA(NNX12AN71G) and the National Science Foundation (MCB0948584and MCB1243792).

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