Báo cáo khoa học: Cellular response to unfolded proteins in the endoplasmic reticulum of plants pptx

In yeast, the UPR increases the folding and degrada-tion capacities of unfolded proteins by inducing the expression of genes related to those capacities [1].. The mammalian UPR is trigge

Cellular response to unfolded proteins in the endoplasmic reticulum of plants

Reiko Urade

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Japan

Introduction

The unfolded protein response (UPR) is a fundamental

system common to unicellular organisms, plants,

ani-mals, and humans, and is conserved in all eukaryotic

cells However, there are differences in the molecular

mechanisms underlying the UPR between organisms

In yeast, the UPR increases the folding and

degrada-tion capacities of unfolded proteins by inducing the

expression of genes related to those capacities [1]

Inos-itol-requiring enzyme-1 (IRE1), an endoplasmic

reticu-lum (ER)-transmembrane protein that is activated by

ER stress, splices basic leucine zipper (bZIP)

transcrip-tion factor HAC1 mRNA in a nonconventranscrip-tional

man-ner [2,3] HAC1 is translated from the spliced mRNA [4–6] and subsequently activates the transcription of a group of genes possessing UPR cis-activating regula-tory elements in their promoter regions [7–9] This pathway was the first example of a protein signal that

is transduced from the ER to the nucleus, and this finding opened the door to investigation of the details

of UPR signaling events

In comparison with that of yeast, the UPR of mam-malian cells is a much more complicated event, in which general attenuation of translation, apoptosis, and folding or degrading of unfolded proteins occurs [10–12] The mammalian UPR is triggered by at least three ER stress sensors, including the mammalian

Keywords

endoplasmic reticulum; ER-associated

degradation; molecular chaperones; protein

folding; quality control of proteins; unfolded

protein response

Correspondence

R Urade, Division of Food Science and

Biotechnology, Graduate School of

Agriculture, Kyoto University, Gokasho, Uji,

Kyoto 611-0011, Japan

Fax: +81 774 38 3757

Tel +81 774 38 3758

E-mail: urade@kais.kyoto-u.ac.jp

(Received 23 November 2006, accepted 22

December 2006)

doi:10.1111/j.1742-4658.2007.05664.x

Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation When protein folding is inhibited because

of mutations or unbalanced ratios of subunits of hetero-oligomeric pro-teins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress As ER stress often disturbs normal cellular functions, sig-nal-transduction pathways are activated in an attempt to maintain the homeostasis of the ER These pathways are collectively referred to as the unfolded protein response (UPR) There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed

Abbreviations

ATF, activating transcription factor; BiP, binding protein; bZIP, basic leucine zipper; eIF2a, initiation factor-2a; ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERSE, ER stress response element; fl-2, floury-2; GFP, green fluorescent protein; GLS, Golgi body localization sequence; GPT, UDP-N-acetylglucosamine–dolichol phosphate N-acetylglucosamine-1-phosphate transferase; IRE1, inositol-requiring enzyme-1; PCD, programmed cell death; PDI, protein disulfide isomerase; PERK, interferon-induced dsRNA-activated protein kinase-related protein; S1P, site-1 protease; S2P, site-2 protease; UGGT, UDP-glucose–glycoprotein glucosyltransferase; UPR, unfolded protein response; UPS, ubiquitin-proteasome system; XBP-1, X-box binding protein 1.

ortholog of yeast IRE1 [13,14], activating transcription

factor (ATF) 6 [15], and interferon-induced

dsRNA-activated protein kinase-related protein (PERK) [16]

IRE1 is activated during ER stress and splices invalid

mRNA, similar to yeast IRE1, into the mature X-box

binding protein 1 (XBP-1) mRNA, a bZIP-like

tran-scription factor [17–20] XBP-1 is translated from the

spliced mRNA and is translocated to the nucleus to

regulate transcription of target genes In addition,

IRE1 independently mediates the rapid degradation of

a specific subset of mRNAs due to their localization

on the ER membrane and to the amino-acid sequence

they encode [21] This response could selectively halt

production of proteins that challenge the ER and

could make available the translocation and folding

machinery for the subsequent remodeling process In

addition, IRE1 forms a trimeric complex with

phos-phorylated tumor necrosis factor receptor-associated

factor 2, apoptosis signal regulating kinase 1 and the

c-Jun N-terminal kinase and subsequently causes cell

death [11,22,23] ATF6 is an ER transmembrane

pro-tein that senses ER stress through its luminal domain,

and then moves to Golgi bodies to be cleaved The

ATF6 cytosolic domain produced as a result of this

cleavage event is released from the Golgi membrane

into the nucleus, where it induces the expression of

tar-get genes [24–28] PERK is an ER transmembrane

pro-tein that senses ER stress through its luminal domain

and phosphorylates a specific serine residue of

transla-tion initiatransla-tion factor-2a (eIF2a), resulting in general

inhibition of translation [16,29] Phosphorylation of

eIF2a also stimulates translation of ATF4 [30], a

bZIP-like transcription factor that induces the

tran-scription of many amino-acid synthetic enzymes,

amino-acid transporters, and antioxidation enzymes

ATF6 and ATF4 also stimulate the transcription of

CHOP, a gene important for apoptotic cell death [31]

It has recently been shown that UPR signaling not

only maintains the homeostasis of the ER, but also

plays an important role in nutritional and

differentia-tion programs in healthy and unstressed yeast and

mammalian cells [11,32,33] Furthermore, organ-specific

UPR signaling pathways have been identified in

mam-malian cells [34–37] These findings suggest that the

UPR functions during normal processes as well as

during emergency situations The UPR pathways act

cooperatively such that the fate of the cell depends on

the balance between the individual UPR pathways

Therefore, disturbance of these functions causes

mal-function of the ER transport machinery and defective

UPR signaling, resulting in diseases such as

neurode-generative disorders, diabetes, and endocrine defects

[11]

The UPR in plants is an important and constantly expanding topic However, study of the plant UPR is

a relatively new field, and its molecular details are only now becoming clear Recent developments in this field will be explored in this review

Transcriptional regulation of UPR genes

The most prominent phenomenon induced by ER stress is transcriptional regulation of UPR genes The induction of genes assumed to be related to the UPR

in plant cells has been reported Binding protein (BiP)

is a representative UPR gene BiP is induced in the presence of drugs that cause ER stress, such as tunica-mycin [38–45] Tunicatunica-mycin inhibits UDP-N-acetyl-glucosamine– dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), such that the initial step

of the biosynthesis of dolichol-linked oligosaccharides

is blocked [46] Treatment with tunicamycin results in the inability of asparagine (N)-linked glycoproteins synthesized in the ER to be glycosylated Transgenic Arabidopsis thaliana plants with a 10-fold higher level

of GPT activity were resistant to tunicamycin at a con-centration that was lethal to control plants [44] Like-wise, transgenic plants grown in the presence of tunicamycin have N-glycosylated proteins, and expres-sion levels of BiP mRNA was lower than in control plants These findings suggest that treatment with tu-nicamycin results in the generation of misfolded or unfolded proteins by inhibiting N-glycosylation and activation of the UPR Transcription of BiP mRNA

is activated by other drugs such as the proline analog azetidine-2-carboxylase, which is incorporated into nascent polypeptides and prevents their folding [47], and dithiothreitol, which inhibits formation of disulfide bonds on nascent polypeptides and prevents their fold-ing [39]

Two comprehensive analyses of the transcriptome of

A thaliana during drug-induced ER stress have been performed using two kinds of DNA microarray meth-ods Martı`nez & Chrispeels [48] performed experiments using an Affymetrix GeneChip with a 8297 probe set (7372 independent genes of the 27 000 protein-coding genes of A thaliana) The UPR was induced by treat-ing Arabidopsis plants with tunicamycin or dithiothrei-tol Fifty-three genes were identified as up-regulated genes under ER stress, whereas 31 genes were identi-fied as down-regulated genes Kamauchi et al [49] analyzed the transcriptome of Arabidopsis UPR genes

by fluid microarray analysis of tunicamycin-treated plantlets Using this method, target genes were cloned from selected fluid microarray beads [50], and 215

up-regulated genes and 17 down-regulated genes were

identified These genes were reanalyzed with functional

DNA microarrays using DNA clones from the fluid

microarray analysis Together, 36 up-regulated genes

and two down-regulated genes in all samples treated

with the three drugs, tunicamycin, dithiothreitol or

azetidine-2-carboxylase were recognized as UPR genes

The up-regulated UPR genes identified by the two

research groups are shown in Table 1, and include ER

chaperones, glycosylation⁄ modification-related

pro-teins, translocon subunits, vesicle transport propro-teins,

and ER-associated degradation (ERAD) proteins

Most of these proteins are orthologs of the genes

iden-tified as being related to the UPR in yeast and

mam-malian cells [1,30,51–54] In addition, genes related to

the regulation of translation (P58IPK) [55] and

apop-tosis (BAX inhibitor 1) [56,57] were also identified as

being up-regulated during the UPR in plants [49,58]

Phospholipid biosynthetic enzymes increase in

expres-sion in the maize (Zea mays) floury-2 (fl-2) mutant

(described below) and soybean (Glycine max)

suspen-sion cultures when treated with tunicamycin [45], and,

in yeast, a number of lipid metabolism-related genes

are up-regulated by ER stress [1] On the other hand,

neither of the DNA microarray analyses of the

Arabidopsis transcriptome described above detected

any up-regulation of lipid metabolism-related genes,

suggesting that additional experiments are needed to

assess if phospholipid metabolism-related genes are

related to the UPR in plant cells

Signal-transduction-related proteins such as protein

kinases and transcription factors are also up-regulated

during the plant UPR WRKY33 and ATAF2 were

identified as repressors of the signal-transduction

path-way activated in response to pathogens [59,60] Zat12

enhances the expression of oxidative-stress and light

stress-response transcripts and plays a central role in

reactive oxygen and abiotic stress signaling [61],

imply-ing that the UPR signal-transduction pathway

con-nects other stress signaling pathways Genes regulated

by other transduction pathways connected with UPR

signal transduction may eventually be identified as

being either up-regulated or down-regulated after

treatment with drugs that induce ER stress The role

of these genes under these circumstances remains to be

elucidated in plants

There are discrepancies in the identification and

ana-lysis of genes down-regulated during ER stress

obtained from the two DNA microarray assays

des-cribed above Thirty-one down-regulated genes were

identified using the Affymetrix GeneChip, and among

them, 29 genes were predicted to encode proteins

con-taining signal peptides Lowering the threshold of

detection from 2.5-fold to 2-fold inhibition increases this amount to 129 independent genes Among these genes, 82% of the encoded proteins have signal pep-tides On the other hand, only two down-regulated genes, vegetative storage proteins Vsp1 and Vsp2, were identified by the fluid microarray method Both of these proteins also have a signal peptide In mamma-lian cells, expression of abundant genes is repressed during ER stress depending on IRE1 but not on XBP-1 Repression of these genes is fast compared with expression changes mediated by XBP-1 Furthermore, functional signal sequences of proteins encoded by down-regulated genes are required for this repression event to occur Taken together, it is possible that IRE1-mediated mRNA degradation occurs during co-translational translocation [21] The fact that more than 80% of the encoded proteins in Arabidopsis with down-regulated expression during ER stress have sig-nal peptides raises the possibility that similar systems may function in plant cells

In both DNA microarray analyses, only the genes that complied with certain restrictive criteria were designated UPR genes, implying that some UPR genes were missed during the analysis as a result of these cri-teria Thus, genes expressed at very low levels might have been unintentionally eliminated from the analysis because of difficulty in assessing differences in their expression levels For example, AtbZIP60, which was not designated a UPR gene by DNA microarray ana-lysis, is induced in response to ER stress as detected

by Northern blot and RT-PCR analyses [62] It is expected that genes identified by the DNA microarray analyses will eventually be confirmed by other methods such as mRNA quantification and promoter analysis

A pivotal role of the UPR is to maintain ER home-ostasis Therefore, the presence of mutated proteins that are unable to fold into their native conformation

in the ER induces the UPR in an effort to restabilize the ER environment Many examples of this phenom-enon have been described in yeast and mammalian cells, and few examples have been found in plants For example, maize high-lysine starchy endosperm (opaque) mutants are characterized by a decrease in the accumu-lation of storage proteins in the ER and by alterations

in protein body morphology in their endosperm The opaquemutants fl-2 and defective endosperm B30 have

a defective signal peptide in the 24-kDa a-zein and the 19-kDa a-zein endosperm storage proteins, respect-ively These mutant proteins are translocated into the lumen of the ER, but remain anchored to the mem-branes through the noncleaved signal peptide [63,64]

A decrease in the expression of a-zein is accompanied

by an increase in the level of b-70, a water-soluble

Table 1 Genes up-regulated during ER stress Data from [48,49] are combined NEM, N-Ethylmaleimide; GST, glutathione S-transferase.

Protein folding

Glycosylation ⁄ modification

At2g41490 UDP-GlcNAc:dolichol phosphate

N-acetylglucosamine-1-phosphate transferase

At4g15550 UDP-glucose indole-3-acetate

b- D -glucosyltransferase

48 Translocation

Protein degradation

Vacuolar

Translation

Vesicle trafficking

PCD

maize BiP ortholog associated with both the ER and

protein bodies [64–70] The increase in maize BiP

mRNA and corresponding protein concentrations

in mutants compared with those of wild-type maize

was endosperm-specific and inversely proportional to

changes in mutant zein synthesis [66] The pattern of

gene expression in normal and the seven opaque

mutants o1, o2, o5, o9, o11, Mc and fl-2, protein

syn-thesis of which is the molecular basis of the mutation,

was assayed by profiling endosperm mRNA transcripts

with an Affymetrix GeneChip containing more than

1400 selected maize gene sequences [71] Compared

with normal maize, alterations in the gene expression

patterns of the opaque mutants were pleiotropic, where

the expressions of BiP, protein disulfide isomerase

(PDI), calreticulin, GRP94 and cyclophilin, and other

physiological stress-related genes were increased in the

opaque mutants The transcriptional response in fl-2

may be induced by the UPR, as the change in the

pattern of gene expression was restricted to the

endo-sperm in which the mutant a-zein was synthesized The

expression pattern of o2 and fl-2 depends on the

molecular basis of the mutation It remains necessary

to evaluate the relationship between the expression

patterns and the molecular basis of each mutation in

the other mutants before a complete understanding of

how these mutants affect ER homeostasis in plants will

be obtained

Signal transduction during the UPR

Transcription of genes related to the UPR is controlled

by the specific transcription factor that binds to the cis-acting regulatory element on the promoter of a UPR gene Many experiments have revealed the details

of the signal-transduction mechanism by which yeast and mammalian cells adapt to ER stress [10,11,72,73]

In yeast, a 22-bp segment in the promoter of KAR2 (yeast BiP) was identified as the first regulatory ele-ment responding to ER stress [7–9], and the sequence CAGCGTG within this 22-bp segment was identified

as the minimal regulatory element and named UPRE (UPR cis-acting regulatory element) HAC1 produced from mRNA spliced by IRE1 binds to the UPRE and induces the transcription of UPR genes [4,5] In mam-malian cells, bZIP-like transcription factors XBP1 [17– 20], ATF6 [15], ATF4 [30], ATF3 [74], CHOP [75], nuclear factor-erythroid 2-related factor 2 [76], OASIS [35], CREB-H [36] and Tisp40 [37] function under ER stress These transcription factors bind to one or more cis-acting regulatory elements and activate or repress the transcription of target genes More than 10 types

Table 1 (Continued ).

Kinase

Transcription factor

Stress protein

Unclassified

At5g09410 Similar to anther ethylene-up-regulated

calmodulin-binding protein ER1

At4g12720 Similar to growth factor protein with

mutT domain

48

a Numbers in parentheses show the number of elements on the promoter.

of cis-acting regulatory elements that respond to ER

stress are known in mammals [11] Among them, ER

stress response element (ERSE) and ERSE-II are

tar-gets for both ATF6 and XBP1 [15,77–79] ATF6 is

constitutively synthesized as a type II transmembrane

protein in the ER [24] When the ER-membrane-bound

precursors of ATF6 are cleaved by the serine protease

site-1 protease (S1P) and the metalloprotease site-2

protease (S2P) in response to ER stress, the N-terminal

halves become soluble transcription factors These

sol-uble factors are translocated into the nucleus and bind

to ERSE and ERSE-II [24–28] ERSE controls the

expression of ER-localized molecular chaperones

[80,81] Transcription from another cis-acting

regula-tory element, XBP1-BS, is entirely controlled by

XBP1, and induces expression of components of the

ERAD system [80,81] In plants, cis-acting regulatory

elements that respond to ER stress have also been

dis-covered The soybean BiP paralog genes gsBIP6 and

gsBIP9 have domains similar to ERSE and ERSE-II

in their 5¢ flanking sequences that are responsive to

treatment with tunicamycin [82] Similarly, a 24-bp

sequence in the 5¢ flanking sequences of Arabidopsis

BiP is crucial for gene induction by tunicamycin [83]

This 24-bp sequence is called P-UPRE and contains

two overlapping elements similar to mammalian

ERSE-II and XBP-BS Putative cis-acting regulatory

sequences similar to ERSE, XBP1-BS, and P-UPRE

are found at high frequencies (> 65%) in the 5¢

flank-ing sequences of the Arabidopsis UPR genes identified

by the DNA microarray analyses (Table 1)

Novel transcription factor AtbZIP60 has been

identi-fied as a member of the plant UPR signal-transduction

pathway To date, every transcription factor related to

the UPR in mammals and yeast is bZIP-like Hence,

Iwata & Koizumi [84] analyzed transcripts of 75

puta-tive bZIP transcription factors in the Arabidopsis

genome Among them, only AtbZIP60, a factor that is

induced by treatment with tunicamycin, dithiothreitol

and azetidine-2-carboxylase, activates transcription

from P-UPRE and ERSE elements The AtbZIP60 gene encodes a predicted type II transmembrane pro-tein of 295 amino acids with an N-terminal bZIP DNA-binding domain, a putative transmembrane domain, and a 56-amino-acid small C-terminal domain (Fig 1A) A truncated form of AtbZIP60 lacking the transmembrane domain (AtbZIP60 DC) fused with green fluorescent protein (GFP) localized to the nuc-leus In other experiments, AtbZIP60 DC clearly acti-vated both P-UPRE and ERSE-like sequences in a dual luciferase assay using protoplasts of cultured tobacco (Nicotiana tabacum) cells Therefore, Atb-ZIP60 is considered to be a transcription factor responding to ER stress, where AtbZIP60 DC induces the expression of AtbZIP60 through ERSE-like sequences present in the promoter of AtbZIP60 In contrast, wild-type AtbZIP60 is unable to activate ERSE-like sequences and P-UPRE, probably because

it is anchored to the membrane This suggests that native AtbZIP60 may be released from the membrane into the cytosol during ER stress to act as a transcrip-tion factor in the nucleus (Fig 2) In the Arabidopsis genome, the At4g20310 gene encodes a membrane pro-tein analogous to S2P, but it remains to be confirmed whether AtbZIP60 is cleaved and released from the membrane during ER stress In addition, no conserved sequence necessary for cleavage by S1P and S2P has been identified near the putative transmembrane domain of AtbZIP60, suggesting that it is possible that AtbZIP60 is released by an unknown intramembrane proteolysis event unique to plant cells

It is not known how AtbZIP60 senses ER stress Two Golgi body localization sequences (GLS1 and GLS2) were identified in the ER-luminal domain of ATF6 [85] ATF6 localizes to the ER through interac-tion between GLS1 and BiP In the absence of BiP, ATF6 is constitutively transported to the Golgi bodies Thus, when unfolded proteins sequester BiP from GLS1 under ER stress, ATF6 is transported into the Golgi body to become a substrate for S1P and S2P

A

B

Fig 1 Comparison of the primary structure

of ATF6 and Arabidopsis bZIP60 (A) and of

yeast IRE1, Arabidopsis IRE1-1 (AtIre1-1)

and Arabidopsis IRE1-2 (AtIre1-2) (B) The

black bar represents the region required for

oligomerization The dotted bars represent

regions that interact with BiP TAD,

Tran-scriptional activation domain; TM,

trans-membrane domain; SP, signal peptide.

Arrows indicate the positions cut by S1P

and S2P.

However, because the luminal domain of AtbZIP60 is

much smaller than that of ATF6 (Fig 1A), it remains

unclear whether it functions as a sensor for ER stress

in a manner similar to ATF6 Investigation into the

cellular localization of AtbZIP60 will probably clarify

these issues

Orthologs of IRE1 have been identified in

Arabidop-sis (AtIre1-1 and AtIre1-2) and rice (Oryza sativa)

(OsIre1) [86–88] Fusion proteins of AtIre1-1, AtIre1-2

or OsIre1 with GFP expressed in tobacco By2 cells

localize to the perinuclear ER The expression patterns

of AtIre1-1 and AtIre1-2 have been examined with

fusion genes of their promoter and a reporter gene

The expression of AtIre1-1 is restricted to certain

tis-sues at specific developmental stages such as the apical

meristem, the leaf margins where vascular bundles end,

the anthers before pollen is formed, the ovules at an

early stage of development, and the cotyledons

imme-diately after germination AtIre1-2 is generally

expressed in plants The C-terminal cytosolic domain

of IRE1ps is conserved among a variety of organisms

(Fig 1B) The C-terminal halves of recombinant

AtIre1-2 and OsIre1 have autophosphorylation

activ-ity When Lys442 of AtIre1-2 was mutated to Ala, this

activity was lost The N-terminal luminal domains of

AtIre1-1, AtIre1-2 and OsIre1 function as ER stress sensors in yeast cells, although the amino-acid sequences of these N-terminal domains are dissimilar from that of yeast IRE1 Thus, when chimeric genes were created by fusing the N-terminal domains of AtIre1-1, AtIre1-2 and OsIre1 with the C-terminal domain of yeast IRE1, and were introduced into a yeast DIre1 mutant, treatment with tunicamycin no longer inhibited growth, and treatments with tunica-mycin or dithiothreitol induced the UPR [86,88] Yeast and mammalian IRE1 function as a sensor to

ER stress through a process involving homodimeriza-tion and autophosphorylahomodimeriza-tion The luminal domain has a BiP-binding site in a region neighboring the transmembrane domain, and dissociation and associ-ation of BiP with this domain regulates the activassoci-ation

of IRE1 [89–91] Thus, IRE1 is inactive when its lumi-nal domain is bound by BiP Upon accumulation of unfolded proteins in the ER, BiP is competitively titra-ted from the luminal domain of IRE1 by the abundant unfolded proteins in the ER lumen, and IRE1 is acti-vated Structural studies of the luminal domains of yeast and human IRE1 show that dimerization of lu-minal domain monomers creates a major histocompati-bility complex-like groove at the interface [92,93]

Fig 2 Model of ER-stress signaling path-ways in plants Question marks indicate incompletely understood relationships.

However, it remains unknown if plant IRE1 orthologs

function as regulators of transcription during ER

stress, but it is possible that BiP plays an important

role in sensing unfolded proteins in the ER, as

overex-pression of BiP in tobacco cells results in a decrease in

the UPR induced by tunicamycin [94]

Plant ER is different from animal ER, in that it is

continuous throughout the entire plant by way of the

plasmodesmata network [95] Certain stress signals,

such as an attack by a pathogen, are transmitted

throughout the plant, giving rise to systematic

induc-tion of specific genes through this continuity of the

ER However, the UPR is restricted to the cells where

the stress was initiated and cannot induce a systemic

response in plants, as transcription of BiP mRNA was

found to be restricted to leaves treated with

tunica-mycin [96]

Enhancing cellular quality control

systems by the UPR

Folding

Folding of nascent polypeptides in cells is not as

effi-cient as was once thought More than 30% of the

nas-cent polypeptides are assumed to be degraded as junk

products before being folded into their proper

confor-mation in the cytosol of animal cells [97] Nascent

polypeptides produced in the ER are presumed to

undergo a similar fate However, folding of

polypep-tides translocated into the ER lumen may fail more

often than that of the polypeptides in the cytosol

because these folding events require more complicated

steps such as glycosylation and⁄ or formation of

disul-fide bonds Therefore, the UPR is considered to be

weakly but constitutively activated and maintains the

homeostasis of the ER even in apparently unstressed

cells In particular, developmental events associated

with high secretory activity are predicted to induce the

UPR [98,99] The quality control of proteins includes

the folding of nascent polypeptide chains into their

native conformation, post-translational modifications

important for proper folding, and the degradation of

misfolded proteins and nonassociated subunit proteins

Enhancement of folding is accompanied by induction

of ER-localized molecular chaperones and foldases

(PDI-related proteins) In Arabidopsis, mRNA of BiP,

the SIL1 homolog, cyclophilin, GRP94 and PDI-related

proteins are up-regulated by the UPR as described

above BiP is best characterized by its role in protein

folding and assembly [100,101] In addition, BiP plays

an essential role in maintaining the permeability

bar-rier of the ER translocon during early stages of protein

translocation [102], targeting misfolded proteins for proteasomal degradation [103,104], sensing ER stress [85,89], and contributing to the ER calcium stores [105] Most of these functions require its ATPase activ-ity, where in the ATP-bound state, BiP is in an ‘open’ form that binds and releases unfolded substrates rap-idly Hydrolysis of ATP drives it to the ADP-bound or

‘closed’ state, thus stabilizing its association with unfolded proteins The release of ADP and the rebind-ing of ATP reopens the substrate-bindrebind-ing domain to release and fold the nascent protein SIL1 is a cochap-erone of BiP and regulates its ATPase cycle by stimu-lating ATP hydrolysis and accelerating the ADP–ATP exchange [106]

Proline can exist in either the cis or trans form in

a polypeptide chain, and its orientation dramati-cally influences the secondary structure of the protein Peptidyl-prolyl-cis-trans isomerases (cyclophilin) survey the status of the proline residues and rearrange them from the cis to the trans form to ensure proper folding

of the nascent polypeptide chains Twenty-nine genes encoding cyclophilin family members are present in the Arabidopsis genome, and five gene products are assumed to be targeted to the ER lumen with N-ter-minal signal peptides [107] Among them, ATCYP20-1

is up-regulated during ER stress, and contains a domain essential for peptidyl-prolyl-cis-trans isomerase activity

Four PDI-related genes are up-regulated during ER stress PDI catalyzes the formation and rearrangement

of disulfide bonds between correct pairs of Cys resi-dues in nascent polypeptide chains in the ER [108] PDI and related proteins are characterized by thiore-doxin motifs within their primary structure [109,110]; Arabidopsis PDI-related proteins, the expression of which is induced during ER stress, have two of these motifs A comprehensive search of the Arabidopsis gen-ome identified 22 orthologs of known PDI-like pro-teins [111] PDI purified from plants or recombinant PDI-related proteins expressed in Escherichia coli have protein disulfide oxidoreductase activity [38,112–116], and their importance in protein folding has been dem-onstrated in rice endosperm [117] In endosperm of rice esp2 mutants lacking PDI, a precursor of the storage protein proglutelin forms aggregates with other storage proteins via interchain disulfide bonds within the

ER lumen, whereas in wild-type rice, proglutelins are processed normally into acidic and basic subunits and accumulate in protein storage vacuoles In soybean cotyledon, PDI-related proteins GmPDIS-1 (an ortho-log of At2g47470) [116] associates with a precursor

of the storage protein glycinin in the ER, suggesting that the PDI-related protein participates in glycinin

folding Yeast and mammalian PDI are activated

by the FAD-dependent oxidases ERO1 and Erv2p

[118–121] Similarly, the Arabidopsis genome encodes

an ERO1 homolog, At2g38960, and an Erv2p

homo-log, At1g15020 or At2g01270, but so far the plant

varieties have not been characterized Mammalian PDI

not only folds polypeptides, but it also aggregates

unfolded proteins via disulfide bonds for retention in

the ER lumen [122], and reduces aggregated proteins

before retro-translocation into the cytosol for

degrada-tion [123] No evidence for the funcdegrada-tion of PDI

pro-teins in plants has been reported

The high-capacity calcium-binding proteins, calnexin

(an ER transmembrane protein) [124,125] and

calreticu-lin (an ER luminal protein) [126,127], are molecular

chaperones in mammalian cells specific for unfolded

N-glycosylated proteins [128] The first step in the

N-glycosylation of a protein is the transfer of a core

glycan Glc3Man9GlucNac2 from a membrane-bound

dolichol phosphate anchor to consensus Asn-X-Ser⁄

Thr residues in the polypeptide chain The glucose

resi-dues on the transferred core glycan are sequentially

trimmed to Glc1Man9GlucNac2 by b-glucosidase I and

b-glucosidase II The monoglucosylated glycan on the

polypeptide chain is trapped by calnexin or calreticulin

to protect it from degradation, resulting in retention of

the polypeptide in the ER for folding [129,130]

The monoglucosylated form of the unfolded protein

shuttles through cycles of deglucosylation by

b-glucosi-dase II and reglucosylation by

UDP-glucose–glycopro-tein glucosyltransferase (UGGT), which preferentially

recognizes unfolded glucosylated glycoproteins [131] This

process is called the calnexin⁄ calreticulin cycle, and is

one arm of the quality control machinery in the

mam-malian ER It is possible that interaction between

monoglucosylated N-glycan with calnexin⁄ calreticulin

functions for the quality control of N-glycosylated

pro-teins in plants, although the calnexin⁄ calreticulin cycle

remains to be elucidated in plants However,

circum-stantial evidence supports the idea that the calnexin⁄

calreticulin cycle is present in plant cells [132] For

example, it has been shown in in vitro translation

sys-tems with wheat germ extract and bean microsomes that

the rate of phaseolin assembly is accelerated when a

glu-cosidase inhibitor is included to stop glucose trimming

of the N-glycan [133] In this system, phaseolin with

par-tially trimmed glycans was unable to assemble into

trim-ers, probably because of being trapped by calnexin or

calreticulin In kaiware radish (Raphanus sativus), the

glucosidase inhibitors castanospermine and

deoxynojiri-mycin suppressed the growth of seedlings by inhibiting

glucose trimming of the N-glycan [134,135], and, in

Arabidopsis, homozygous deletion of b-glucosidase I by

T-DNA tagging is lethal [136] In potato, curled leaves and low yields have been reported when expression of the b-glucosidase II gene MAL1 was knocked-down by antisense RNA [137] Furthermore, the knock-down of MAL1caused an increase in the expression of BiP, sug-gesting the presence of ER stress In Arabidopsis rsw3, a temperature-sensitive mutant of the b-glucosidase II b-subunit, some morphological abnormalities and growth impairments were observed [138] As trimming glucose residues of N-glycan by b-glucosidase I and b-glucosidase II is a prerequisite for modification of the ER-type glycan to the complex glycan in Golgi bodies,

it is possible that the impairment of this process is responsible for the adverse effects on plant morphology However, this explanation may be unlikely, as neither growth inhibition nor reproduction defects have been observed in Arabidopsis mutants defective in GlcNAc-transferase I, which catalyzes the first modification reac-tion to the complex-type glycan [139]

UDP-glucose, the substrate for re-glucosylation of N-glycan by UGGT, is synthesized in the cytosol, indi-cating that a UDP-glucose transporter would be required for the calnexin⁄ calreticulin cycle AtUTr1 from Arabidopsis is an ER-localized membrane pro-tein, the expression of which is induced by treatment with dithiothreitol [140], and is recognized as a UDP galactose⁄ glucose transporter [141] In addition, up-regulation of the ER chaperones, BiP and calnexin, has been observed in an AtUTr1 insertional mutant, suggesting that these plants may constitutively activate the UPR Taken together, it is possible that the

calnex-in⁄ calreticulin cycle discriminates between folded and unfolded glycoproteins in plant cells In mammalian cells, the recognition of the unfolded glycoproteins by calnexin⁄ calreticulin is coupled with the formation of disulfide bonds, where the PDI-related thiol-oxidore-ductase, ER-60⁄ ERp57, interacts with the P domain of calnexin or calreticulin to fold N-glycosylated proteins [142–144] The amino-acid sequence of the P domain

of plant calnexin and calreticulin is highly conserved compared with that of its mammalian counterparts [145,146] However, it is not known whether plant calnexin or calreticulin cooperates with any plant PDI-related oxidoreductase to form disulfide bonds in N-glycosylated proteins

Degradation of unfolded proteins Unfolded proteins generated in the rough ER are predominantly degraded by ERAD in yeast and mammalian cells [147], requiring that the unfolded polypeptides be transported across the ER membrane into the cytosol via a translocon located on the ER

membrane [148] to be degraded by the cytoplasmic

ubiquitin-proteasome system (UPS) [149]

In plants, misfolded storage proteins generated in

the ER are degraded by an unidentified system

[150,151] However, it has been proposed that both

ERAD and a vacuolar system may degrade the

unfol-ded proteins generated in the rough ER, although the

details of this mechanism have not been established

In plants, UPS-dependent and UPS-independent

ERAD-like degradation have been observed Ricin is a

heterodimeric ribosome-inactivating protein that

accu-mulates in castor beans (Ricinus communis) The

mature ricin comprises a catalytic A chain and a B

chain linked by a single disulfide bond The

ER-tar-geted A chain is degraded by a pathway that closely

resembles ERAD when expressed in tobacco

proto-plasts in the absence of a B chain [152] The

degrada-tion of ricin A chain is brefeldin A-insensitive and is

inhibited by the proteasome inhibitor clasto-lactacystin

b-lactone, resulting in the accumulation of ricin

A chains These stabilized ricin A chains are partly

deglycosylated by a peptide–N-glycanase-like activity

Taken together, these results indicate that the ricin A

chain behaves as a substrate of the ERAD where it is

exported into the cytosol, deglycosylated, and

degra-ded by the proteasome [153,154] A mutant of barley

(Hordeum vulgare) mildew resistance O protein-1 is

also degraded by UPS-dependent ERAD in plants

[155] Individual mutant mildew resistance O protein-1

proteins with single amino-acid substitutions in its

seven-transmembrane domain exhibit markedly

reduced half-lives, are polyubiquitinated, and can be

stabilized through inhibition of proteasome activity

When the mutant mildew resistance O protein-1 is

transfected into Arabidopsis plants previously transfected

with dominant negative mutants of the putative AAA

ATPase AtCDC48A⁄ p97 (a component of the ERAD

machinery) [156,157], the degradation of the mutant

mildew resistance O protein-1 is impaired This

strongly suggests that mildew resistance O protein-1 is

an endogenous substrate of a UPS-dependent

ERAD-related quality control mechanism in plants

In plants, several misfolded proteins are translocated

across the ER membrane to the cytosol and degraded

by an unknown UPS-independent system The

C-ter-minal extension mutant of phaseolin transfected into

tobacco protoplasts is degraded very rapidly in a

bre-feldin A- and proteasome inhibitor-insensitive manner

[158], suggesting that it is performed in a pre-Golgi

compartment, probably in the cytosol Likewise, when

both endogenous and recombinant cell wall invertases

are synthesized without their N-glycans in BY2

tobacco cells, they both degrade very rapidly [159]

This degradation does not occur in an acidic com-partment and is also insensitive to brefeldin A and proteasome inhibitor Furthermore, a fusion protein consisting of misfolded N-terminally truncated calreti-culin with GFP is also retrotranslocated from the ER lumen to the cytosol and is subsequently degraded [160,161] The dislocated fusion proteins accumulate in the nucleoplasm in a microtubule-dependent manner and are degraded very slowly by an unknown UPS-independent system These UPS-UPS-independent ERAD-like degradations are unique in plants However, any underlying molecular mechanism of the system remains unknown

Some genes relevant to the translocation of misfolded proteins across the ER membrane into the cytosol are induced during ER stress in Arabidopsis (Table 1) SEC61 subunits form the specific translocon required for retro-translocation of misfolded polypeptides [162] Stress-associated ER protein 1 (SERP1)⁄ Ribosome-associated membrane protein 4 (RAMP4) orthologs are also up-regulated during ER stress SERP1⁄ RAMP4 interacts with the SEC61 a-subunit, the SEC61 b-sub-unit, and calnexin [163,164] This complex stabilizes membrane proteins in the ER membrane through a translocational pausing mechanism [165] P58IPK was previously implicated in translational control (described below) Recently, the novel role of mammalian P58IPK

in the control of the translocation of newly synthesized polypeptides to the ER lumen was reported by Oyadomari et al [166] P58IPK associates with SEC61, recruits HSP70 chaperones to the cytosolic face of SEC61 and associates with translocating polypeptides during ER stress In P58IPK-knockout mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress On the basis of these results, P58IPK is thought to be a key mediator

of cotranslocational ER protein degradation, and probably contributes to ER homeostasis in stressed cells

Genes that stimulate vesicle transport from the ER

to the cis-Golgi are induced during ER stress in Ara-bidopsis (Table 1) Among them, EMP24, SAR1B and SEC23 are shown to make a complex with subunits of the COPII coat, which are key molecules for export of proteins from the ER, and promote transport of newly synthesized proteins from the ER into ER subdomains

or Golgi in yeast [167–170] Newly synthesized proteins that do not fold correctly in the ER are targeted for ERAD through distinct sorting mechanisms; soluble luminal ERAD substrates require ER–Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER [169] Retained transmembrane proteins are often