Recent advances in plant membrane-bound transcription factor research:Emphasis on intracellular movement

Pil Joon Seo

1Department of Bioactive Material Sciences,Chonbuk National University,Jeonju 561-756,Korea,2Research Center of Bioactive Materials,Chonbuk National University,Jeonju 561-756,Korea,3Department of Chemistry and Research Institute of Physics and Chemistry,Chonbuk National University,Jeonju 561-756,Korea.

INTRODUCTION

Regulation of gene expression is a fundamental process of modulating plant growth,developmental processes,and environmental adaptation responses(Hagen and Guilfoyle 2002;Singh et al.2002;Mele and Hake 2003;Shinozaki et al.2003;Yang et al.2012).Gene transcription is regulated primarily by transcription factors,which perceive internal and external cues(Zhang 2003;Chen and Zhu 2004).Accordingly,transcription factors constitute a critical component of gene regulatory networks,and thus understanding the molecular mechanisms by which transcription factor activity is regulated can provide biological insight into the molecular and physiological changes that occur in plants(Yanagisawa 1998).

Transcription factor activities are coordinately regulated at multiple steps in diverse cellular signaling networks for optimal growth and survival under given growth conditions,including at the levels of transcription,post-transcriptional RNA processing,translation,post-translational modification,and protein turnover(Shinozaki et al.2003;Vazquez 2006;Yamaguchi-Shinozaki and Shinozaki 2006;Xu et al.2007;Seo et al.2011).

Over the last decade,a novel transcriptional regulation mechanism,so-called proteolytic processing of membranebound transcription factors(MTFs),has been extensively investigated(Chen et al.2008;Seo et al.2008).MTFs are synthesized in the cytoplasm as full-size proteins with a transmembrane domain(TM)and are rapidly transported to cellular membranes,including nuclear-,endoplasmic reticulum(ER)-,and plasma membrane,where they are stored in dormant form.Upon exposure to developmental and environmental cues,MTFs are proteolytically processed and liberated from the membrane(Kim et al.2006,2008;Liu et al.2008;Seo et al.2010a,2010b).In eukaryotes,processing of MTFs is mediated primarily by two representative mechanisms:regulated intramembrane proteolysis(RIP)and regulated ubiquitin/proteasome-dependent processing(RUP)(Hoppe et al.2001;Seo et al.2008).Finally,after proteolytic processing,MTFs are translocated to the nucleus,where they act on genomic DNA.Interestingly,it has recently been proposed that alternative splicing can also function to release MTFs from the membrane by removing the transmembrane domain(Li et al.2012;Lu et al.2012;Takahashi et al.2012),which has emerged as a novel mechanism of MTF activation,in addition to proteolytic processing.

In plants,investigation of MTFs has focused on two major transcription factor families,namely,(NAC)and basic Leucine Zipper(bZIP)transcription factors(Chen et al.2008;Seo et al.2008).However,recent extensive genome-wide analyses have revealed that Arabidopsis has a much larger number of MTFs in its genome than we expected.Over 100 transcription factors are estimated to be anchored in the cellular membranes(Bateman et al.2009).To date,a total of eight NAC,three bZIP,one MYB,and one PHD transcription factors have been functionally characterized(Table 1).These MTFs are involved in diverse aspects of plant development,including cell division(Kim et al.2006),floral transition(Kim et al.2007a;Li et al.2010),sugar signaling(Li et al.2011),chloroplast development(Sun et al.2011),root development(Slabaugh et al.2011),hormone signaling(Park et al.2011),and seed germination(Kim et al.2008).

Notably,the majority of plant MTFs play significant roles in mediating responses to environmental stress(Liu et al.2007b,2008;Seo et al.2010a,2010b;Klein et al.2012).Physical and chemical properties of membranes are intimately associated with environmental changes,as exemplified by temperaturedependent changes in membrane fluidity(Orvar et al.2000;Degenkolbe et al.2012).Given that membrane reorganization is a primary response to environmental changes(Orvar et al.2000;Paulucci et al.2011;Verelst et al.2013),MTFs and/or MTF processing machineries sense the modifications and ensure rapid transcriptional responses under stressful conditions.

While the roles of plant MTFs are diverse,they share a high degree of structural and mechanistic similarities.Most of the MTFs characterized in Arabidopsis are type II membrane proteins in that the N-terminus of which faces the cytoplasm with single-pass TM domain(Seo et al.2008).The majority of plant MTFs are cleaved by intramembrane proteases,further supporting a conserved feature of plant MTF actions(Chen et al.2008;Seo et al.2008).Accordingly,a few common proteases involved in bZIP MTF processing have been identified(Liu et al.2007b;Srivastava et al.2012),although many of the responsible proteases still remain to be elucidated.The mechanism of proteolytic activation of MTFs is also relevant in other plant species,including rice,soybean and maize,indicating that MTF is a representative way to modulate transcription factor activity in plants(Kim et al.2010;Le et al.2011).

A long-standing question in plant MTF research is whether proteolytic activation is sufficient to mediate their transport to desirable subcellular compartments.Molecular mechanisms underlying intracellular movement of MTFs have only recently been proposed.Regulation of intracellular movement provides an additional layer of complexity of MTF regulation,expanding signal transduction schemes.In this review,we summarize recent advances in functional characterization of MTFs in plants and discuss molecular mechanisms underlying MTF activation,with an emphasis on intracellular movement.Many excellent recent reviews(Chen et al.2008;Seo et al.2008;Liu and Howell 2010b;Iwata and Koizumi 2012)cover in detail other particular aspects of structural organization and function of MTFs that are not fully addressed in this review.Readers are encouraged to consult them.

Table 1.Functions of membrane-bound transcription factors(MTFs)in Arabidopsis

MTFS IN PLANT DEVELOPMENT

The NAC WITH TRANSMEMBRANE MOTIF 1(NTM1)transcription factor is a founding member of NAC MTFs.NTM1 is anchored in the nuclear membrane as a dormant form and is proteolytically processed through the action of a cysteine protease in response to cytokinin treatment(Kim et al.2006).Activated NTM1 is transported to the nucleus and subsequently induces a subset of cyclin-dependent kinase(CDK)inhibitor/KIP-RELATED PROTEIN(KRP)genes that negatively regulate the cell cycle(Wang et al.2000;Verkest et al.2005).The NTM1 protein constitutes cytokinin signaling loops and balances cell division in Arabidopsis.

A plant homeodomain(PHD)transcription factor,PHDTYPE TRANSCRIPTION FACTOR WITH TRANSMEMBRANE DOMAINS(PTM),relays chloroplast signals to the nucleus(Sun et al.2011).While chloroplasts have their own genome,more than 90%of chloroplast proteins are encoded in the nuclear genome(Keegstra and Cline 1999).Hence,retrograde signals from chloroplasts to the nucleus are important for ensuring chloroplast development and maintenance.The PTM transcription factor is bound to chloroplast envelopes and monitors physiological and metabolic states of chloroplasts.Upon the retrograde signals,such as photo-induced damages,PTM is proteolytically processed and translocated to the nucleus(Sun et al.2011).In the nucleus,PTM regulates the ABA INSENSITIVE 4(ABI4)gene by binding directly to the gene promoter(Sun et al.2011),which serves as nuclear master regulator transmitting retrograde signals.This intriguing example sheds light on how organelle-to-organelle communication is coordinated.

A membrane-anchored member of plant-specific R2R3 MYB transcription factor family appears to control root hair development.The membrane-anchored MYB(maMYB)protein contains two TM domains located in the N-and C-termini,respectively,and is associated with the ER membrane(Slabaugh et al.2011).In plant cells,truncated proteins,which lack TM domains but retain the transcription factor domain of maMYB,are clearly migrated to the nucleus(Slabaugh et al.2011).The maMYB gene is transcriptionally induced by exogenous auxin,and maMYB-deficient mutants have defects in root hair elongation.While additional investigation is needed to define the signaling pathway mediated by maMYB,it is highly plausible that maMYB is involved in auxin-mediated regulation of root hair development.

MTFS IN PLANT RESPONSES TO ENVIRONMENTAL STRESS

The NTM2 transcription factor,which shares the highest level of homology with NTM1,integrates salt signaling into the developmental program(Park et al.2011).Seed germination is a plant-specific developmental transition that determines embryonic growth.Multiple internal and external signals,such as gibberellic acid(GA),abscisic acid(ABA),and high salinity,have to be monitored in order to determine the proper time to germinate(Seo et al.2009).The NTM2 gene plays a key role in this process.NTM2 is transcriptionally induced by high salinity and regulates sensitivity to high salt.The NTM2-deficient ntm2-1 mutants are less sensitive to high salt not only at the seedling stage,but also during germination(Park et al.2011).Concomitantly,NTM2 is implicated in modulating auxin signaling via the INDOLE-3-ACETIC ACID 30(IAA30)gene,thereby linking salt signals to auxin signaling.Consistent with this observation,a plant’s sensitivity to high salt is increased by exogenous application of auxin,and the effect of which depends on NTM2(Park et al.2011).

A genome-wide search of Arabidopsis MTFs that are structurally similar to NTM1 resulted in identification of 11 additional NAC MTFs,namely2007b).NTLs possess single α-helical TM domain in the C-terminus and have a conserved NAC DNA-binding domain in their N-terminus.Functional characterization of NTLs has further supported a variety of physiological roles in Arabidopsis.

A considerable number of MTFs are associated with stress responses.The NTL1/ANAC13 gene(At1g32870)is supposed to be responsible for ultraviolet(UV)-light responses in Arabidopsis(Safrany et al.2008).Transcription of NTL1 is induced upon exposure to UV-B,which is likely mediated through a COP1-independent pathway.Promoter deletion analysis has revealed that two cis-regulatory elements,the MYB recognition element(-AACCTT-)and UVBox(CAAG),are required for full induction of NTL1(Safrany et al.2008).While its molecular function is currently elusive,it is highly probable that NTL1 participates in the UV signaling pathway.

A couple of MTFs have been shown to mediate osmotic stress regulation of leaf senescence.The drought stressinducible NTL4 protein triggers reactive oxygen species(ROS)production by binding directly to gene promoters encoding ROS biosynthetic enzymes AtrbohC and AtrbohE(Lee et al.2012).The NTL4-deficient mutants exhibit relatively low levels of ROS accumulation,enhanced drought tolerance,and prolonged leaf longevity under water-limiting conditions.In contrast,ectopic expression of NTL4 results in programmed cell death(PCD)even under normal growth conditions.Altogether,NTL4 contributes to plant fitness and adaptability under adverse growth conditions.NTL9 also underlies osmotic stress regulation of leaf senescence(Yoon et al.2008).Proteolytic processing of the NTL9 protein is induced by osmotic stress,and activated nuclear NTL9 subsequently regulates expression of a subset of senescence-associated genes(SAGs)in the nucleus,thereby establishing crosstalk between stress signaling and internal developmental programs.

Resistance to invading pathogens is elicited at low temperatures and would likely be an evolutionary trace to confer resistance to snow mold(Seo et al.2010a).NTL6 is regarded as a cold sensor that regulates temperaturedependent pathogen resistance in Arabidopsis(Seo et al.2010a).NTL6 is anchored in the plasma membrane and is released from the membrane by a metalloprotease upon exposure to low temperatures(Seo et al.2010b).In particular,cold-induced changes in membrane fluidity are recognized either directly by NTL6 or a yet to be identified NTL6 processing factor.Following proteolytic processing,NTL6 is localized to the nucleus,where it regulates pathogenesisrelated(PR)genes by binding directly to their promoters(Seo et al.2010a),conferring temperature-dependent pathogen resistance.NTL6 is the first exquisite example showing that physico-chemical properties of the membrane are associated with MTF activation.Since membrane properties are rapidly influenced by environmental fluctuation(Orvar et al.2000;Paulucci et al.2011;Verelst et al.2013),it is likely that MTFs enable plants to take advantage in order to respond promptly under stressful conditions.

NTL8 also incorporates environmental signals into a developmental program.Seed germination is considerably delayed under high salinity as a result of altered GA and ABA levels(Seo et al.2009).The NTL8 protein is proteolytically activated in the presence of high salt.The NTL8 liberation is followed by repression of the GA3 oxidase 1(GA3ox1)gene in an ABA-independent manner(Kim et al.2008).Consistently,NTL8-deficient mutants are insensitive to high salt and GA biosynthetic inhibitor paclabutrazol(PAC),indicating a role in GA regulation of salt signaling in seed germination.Moreover,NTL8 plays an additional role in floral transition.Transgenic plants overproducing nuclear NTL8 exhibit late flowering by suppressing FLOWERING LOCUS T(FT)expression(Kim et al.2007a).These observations are also in accordance with salt-induced proteolysis of NTL8.High salinity inhibits floral transition via an NTL8-FT module,ensuring adaptation fitness under such conditions.

The membrane-tethered ANAC089 transcription factor has been implicated in a variety of cellular pathways,including sugar signaling,ROS signaling,and floral transition(Li et al.2010,2011).ANAC089 is anchored in the trans-Golgi network as well as ER membrane and is liberated from the membranes in response to reducing agent treatments(Klein et al.2012).One of the best-known targets of ANAC089 is stromal ascorbate peroxidase (sAPX),which functions in the chloroplast antioxidant defense system(Giacomelli et al.2007).Since ANAC089 is a transcriptional repressor,it functions to lower the accumulation of sAPX transcripts under reducing conditions,forming a negative retrograde loop(Klein et al.2012).In addition to the role of ANAC089 in ROS defense,transgenic plants overproducing truncated ANAC089,which lacks a transmembrane domain in its C-terminus,exhibit fructose insensitivity as well as delayed floral transition(Li et al.2010,2011),presumably suggesting the presence of signaling crosstalk among ROS signaling,sugar metabolism,and developmental transition.

MTFS IN ER STRESS RESPONSE

Several bZIP transcription factors are attached mainly in the ER membrane and are involved in the ER stress responses caused by unfolded or misfolded proteins(Tajima et al.2008;Urade 2009;Iwata and Koizumi 2012).The bZIP MTFs screen the protein folding in the secretary pathway and are proteolytically processed to be translocated to the nucleus when improperly folded proteins accumulate in the ER(Liu and Howell 2010b;Iwata and Koizumi 2012).It is considered a way of sensing unfolded and/or misfolded proteins in the ER in order to initiate the unfolded protein response(UPR).The UPR is also closely associated with environmental stress resistance.When harsh environmental fluctuation is applied,unfolded and/or misfolded proteins noticeably accumulate probably owing to disturbed folding environment in the ER(Liu and Howell 2010b).Consistently,BINDING PROTEINs(BiPs),the most abundant chaperone proteins in the ER,comprehensively accumulate in response to environmental stresses,such as temperature extremes and drought(Anderson et al.1994;Valente et al.2009).

bZIP60 is a founding member of the bZIP MTFs involved in the ER stress response(Iwata and Koizumi 2005).bZIP60 is bound to the ER membrane and is proteolytically cleaved upon a potent stimulus of ER stress.The C-terminal domain of bZIP60 is likely responsible for sensing ER stress in the ER lumen,whereas the N-terminal domain is responsible for DNA binding and transcriptional regulation(Iwata et al.2008).Thus,the N-terminal domain is translocated to the nucleus following proteolytic activation,where it subsequently regulates ER stress-responsive genes(Iwata et al.2008,2009).Notably,cleavage of bZIP60 is independent of SITE-1 PROTEASE(S1P)and S2P,unlike other bZIP MTFs involved in the ER stress response(Iwata et al.2008).

The ER-residing bZIP28 protein is a well-studied MTF and serves as a representative signaling regulator of UPR(Liu et al.2007a).Pharmacological chemicals eliciting ER stress,such as tunicamycin and dithiothreitol(DTT),induce processing of bZIP28 to mediate its relocalization to the nucleus(Liu et al.2007a).The sequential actions of two proteases in the Golgi,namely,subtilisin-like serine protease S1P and metalloprotease S2P,are required for bZIP28 processing.Constitutive expression of a truncated form of bZIP28 containing the N-terminal cytoplasmic domain leads to upregulation of ER stressresponsive genes,including BiP1-3,PROTEIN DISULFIDE ISOMERASE-LIKE PROTEIN(PDIL),CALRETICULIN-1(CRT1),and CALNEXIN-1(CNX1),most likely through direct binding to P-UPRE and ER stress-responsive element(ERSE)(Tajima et al.2008).

The bZIP17 transcription factor plays a key role in ER stress and salt stress(Liu et al.2007b,2008).bZIP17 is proteolytically cleaved by salt stress,and the S1P protease is required for membrane release of bZIP17 under high salinity(Liu et al.2007b).Following proteolytic cleavage,nuclear bZIP17 is translocated to the nucleus,where it regulates expression of salt stress-responsive genes,such as ARABIDOPSIS THALIANA HOMEOBOX 7(ATHB-7).Consistent with these observations,mutants that have defects in either S1P or bZIP17 are hypersensitive to high salinity.In addition,it has been recently demonstrated that bZIP17 is also regulated by ER stress inducers,such as tunicamycin,further supporting the extensive crosstalk between the ER and environmental stress signaling(Che et al.2010).

While bZIP MTFs share biological functions as regulators of the UPR,they perceive different environmental stimuli.For example,the bZIP28 transcription factor is responsible for heat stress-induced UPR responses(Gao et al.2008).The bZIP17 MTF is important for proper folding in high salinity and at high temperature(Liu et al.2007b;Tajima et al.2008).Both bZIP17 and bZIP28 also activate BR signaling in response to environmental stresses,thereby linking BR signaling to stress acclimation(Che et al.2010).bZIP60 responds to salt stress and confers resistance to high salinity(Fujita et al.2007).Together,these observations imply that responses to ER stress and environmental stresses have evolved as coregulated systems,and that bZIP MTFs are the major players in these signaling networks(Liu and Howell 2010b).

PROTEOLYTIC PROCESSING OF PLANT MTFS

Despite the diverse functions of MTFs in plants,they share conserved regulatory schemes.The first regulatory step that MTFs encounter is activation by means of proteolytic processing.It facilitates liberation of MTFs from the membrane,and is regarded as a rate-limiting step to initiate transcriptional signaling cascades from the membrane.Hence,early research on MTFs has focused on how MTFs are proteolytically processed and which proteases or E3 ligases are responsible for this process.

The RIP mechanism is pervasive in the majority of plant MTFs that have been identified thus far.Therefore,extensive studies have focused on genome-wide searches for responsible proteases.bZIP MTFs are mainly processed via the sequential action of the S1P and S2P proteases(Liu et al.2007a),as observed in mammals(Ye et al.2000).However,not all bZIP MTFs undergo conventional two-step processing.For instance,neither S1P nor S2P are required for activation of bZIP60(Iwata et al.2008).

Proteases that are responsible for processing of NAC MTFs remain obscure.Since NACs are a plant-specific transcription factor family,plant-specific proteases and/or unique processing mechanisms may be involved.To date,only a few examples have been illustrated.Processing of NTM1 is modulated by a calpain protease(Kim et al.2006).As-yet-unidentified metalloproteases are supposed to be involved in NTL6 and NTL8 processing under cold and high salt conditions,respectively(Kim et al.2008;Seo et al.2010b).Further investigation is needed to clarify how processing of NTL,and other MTF proteins,are regulated.

ALTERNATIVE SPLICING-ASSISTED MTF ACTIVATION

Proteolytic processing was once regarded as the sole mechanism of MTF activation in plants.Surprisingly,it has been recently shown that alternative splicing also contributes to activation of MTFs(Li et al.2012;Lu et al.2012).Multiple reorganization of exon–intron composition enables living organisms to produce a number of proteins over a limited number of genes.Indeed,several MTF genes undergo alternative splicing and produce splice variants that retain the nuclear transcription factor domain but lack transmembrane domains.This observation indicates that MTF activation is regulated at multiple steps,at the very least at posttranscriptional and post-translational levels(Figure 1),to ensure proper action in response to external and internal stimuli.

Figure 1.Schematic diagram of intracellular movement of NAM/ATAF1/2/CUC2(NAC)and basic Leucine Zipper(bZIP)membranebound transcription factors(MTFs)(A)Nuclear import of NTL6.NTL6 is localized in the plasma membrane under normal conditions.Under stress conditions,the NTL6 protein is processed by an as-yet-unidentified intramembrane protease and transported to the nucleus.The SnRK2.8 kinase is responsible for phosphorylation of NTL6 and facilitates its nuclear import.N,nucleus;P,phosphorylation.(B)Intracellular movement of bZIP28 and bZIP60.Alternative splicing of bZIP60 gene produces two isoforms,one encoding endoplasmic reticulum(ER)membrane-localized dormant transcription factor and the other encoding nuclear transcription factor.Upon accumulation of unfolded proteins in the ER lumen,IRE1 catalyzes cytoplasmic splicing of bZIP60.ER-resident bZIP28 transcription factor is translocated to the Golgi upon ER stress through interaction with Sar1b and Sec12.In the Golgi,sequential proteolysis mediated by S1P and S2P releases processed form of bZIP28.The N-terminal portion moves into the nucleus and recruits nuclear factor Y subunits to regulate ER stress-responsive genes.

The INOSITOL-REQUIRING ENZYME 1(IRE1)protein that senses ER stress and triggers unconventional cytoplasmic splicing has been shown to be responsible for the UPR in yeast and animals(Ron and Walter 2007).The IRE1 splicing factor constitutes an important UPR signaling along with MTFs and protein kinase-like ER kinases(PERKs).Arabidopsis has two IRE1 homologs,whereas rice only contains one member in its genome(Koizumi et al.2001;Okushima et al.2002).Notably,IRE1 splices bZIP60 mRNA and removes a 23-nucleotide intron,producing an active transcription factor in Arabidopsis(Nagashima et al.2011;Iwata and Koizumi 2012).This molecular mechanism is also conserved in monocot plants.OsbZIP50,an ortholog of AtbZIP60,is spliced by OsIRE1 in response to ER stress(Li et al.2012).Moreover,ZmbZIP60 undergoes alternative splicing upon ER stress(Lu et al.2012),producing a splice variant that is truncated in their C-terminus having TM domain and thus is targeted to the nucleus.Together,membrane-tethered MTFs are activated by at least two independent mechanisms:proteolytic processing and alternative splicing.While RIP guarantees a prompt response to external stimuli as exemplified by bZIP17 and bZIP28,which are activated by S1P and S2P(Liu et al.2007a,2007b),alternative splicing-assisted MTF activation is responsible for the late response to internal and external stimuli,because a translation step is required following mRNA metabolism(Nagashima et al.2011;Iwata and Koizumi 2012).

INTRACELLULAR MOVEMENT OF MTFS

To act as transcription factors,MTFs must be relocalized to the nucleus.Thus,a long-standing question was whether the liberation of MTFs from the cellular membrane is sufficient for their nuclear import.Three major research groups have respectively investigated intracellular movement of NAC and bZIP MTFs,and have made significant progress towards answering this question(Kim et al.2012;Srivastava et al.2012;Sun et al.2013).

NAC MTFs seem to be processed by a single proteolysis step at the membrane(Seo et al.2008;Kim et al.2012).In this case,nuclear import following proteolytic processing is a key molecular event to be elucidated.In contrast,ER-resident bZIP MTFs go through two-step sequential processing that takes place in the Golgi(Liu et al.2007a).Thus,the mechanism of how intracellular movements,ER-to-Golgi and Golgi-to-nucleus,are mediated remains a fundamental question in the function of bZIP MTF.A representative working model of individual MTF groups has recently been proposed.While not all MTFs are regulated under the same model,it nevertheless provides insight into how intracellular movement of MTFs is organized,as well as the biological relevance of additional layers of MTF regulation.

Intracellular movement of bZIP28

The bZIP28 transcription factor is synthesized in the cytoplasm and anchored in the ER membrane(Liu et al.2007a).BiP proteins retain bZIP28 in the ER lumen via interaction with the C-terminal tail of bZIP28(Srivastava et al.2013).Upon exposure to ER stress,BiPs dissociate from bZIP28 with ADP/ATP exchange,and thereby allowing bZIP28 to translocate to the Golgi,the site of proteolytic processing.In accordance with this,transgenic plants overexpressing either BiP1 or BiP3 show ER retention of bZIP28 even under ER stress conditions.In contrast,a certain extent of bZIP28 is liberated from ER to nucleus under normal conditions in bip-deficient mutants(Srivastava et al.2013).Dissociation of BiP from bZIP28 is independent of exit from the ER and proteolytic cleavage in the Golgi.The bZIP28 proteins harboring mutations in the region responsible for its liberation from the ER still dissociate from BiPs following tunicamycin treatment(Srivastava et al.2013),indicating that additional factors are required for subsequent regulation(Figure 1).

Dibasic amino acid sequences are required for translocation of MTFs from the ER to the Golgi(Srivastava et al.2012).This region is critical for interaction with Sec12 and the Sar1b small GTPase,components of the coat protein complex II(COPII)machinery that directly interacts with cargo and facilitates its transport from the ER to Golgi(Marti et al.2010).It is noteworthy that the COPII machinery also guides intracellular movement of the bZIP28 MTF.Dibasic motifs(KK311 and KK320;KK positions at position 311 and 320 residues),which lie proximal to the transmembrane domain on the cytosolic side,are required for ER-to-Golgi transport in response to ER stress(Srivastava et al.2012).Lumen-facing C-terminal domain also contributes to ER-to-Golgi transport(Sun et al.2013).Expression of serial deletions on bZIP28 lumen-facing domain has revealed that LD3 truncated protein(Δ560–675 residues),which is 17 amino acids shorter from S1P cleavage site (RRIL576),fails ER-to-Golgi translocation.Accordingly,ER stress-responsive genes are not induced by tunicamycin treatment in LD3-complemented bzip28bzip60 double mutant plants(Sun et al.2013).Given that LD2 protein(Δ577–675 residues)is functionally compatible to ER-to-Golgi movement,the protein region of 560–576 residues may contain an additional Golgi localization signal.In addition,LD4 protein(Δ426–675 residues)is constitutively localized in Golgi,demonstrating that the region of residues 426–559 is responsible for ER retention.

The helix-breaking residue(G329)in the transmembrane domain is crucial for proteolytic processing of bZIP28,which is mediated by S1P and S2P(Srivastava et al.2012).The G329 residue helps to induce conformational changes and release the transcription factor from the Golgi.When the G329 residue is mutated with a G329A substitution,Golgi localization of the protein is retained even under ER stress conditions(Srivastava et al.2012).

A heterotrimeric transcriptional protein complex is formed in the nucleus,which consists of NF-YB3/NF-YC2/NF-YA4,and bZIP28,and binds to ERSE-I DNA elements(CCAAT-N10-CACG).Accordingly,reducing agent treatment results in upregulation of NF-YC2 and translocation of NF-YB3 from the cytoplasm to the nucleus,concomitant with nuclear import of bZIP28,thereby coordinating regulation of UPR-associated genes(Liu and Howell 2010a).In addition,bZIP28 also forms homo-and hetero-dimers with bZIP17,bZIP49,and bZIP60,further expanding the combinatorial diversity and regulatory repertoires(Liu and Howell 2010a).While it is too premature to conclude,extensive interaction with nuclear proteins likely facilitates nuclear relocalization of MTFs and allows them to be retained in the nucleus.

Nuclear import of NTL6 transcription factor

The plasma membrane-localized NTL6 protein is proteolytically processed in response to ABA and cold stress(Seo and Park 2010;Seo et al.2010a).Single proteolysis is implicated in NTL6 activation.After liberation from the plasma membrane,NTL6 is translocated to the nucleus,where it regulates target genes,such as COLD-REGULATED 15a(COR15a)and PR genes(Seo et al.2010a;Kim et al.2012).

Through extensive screen to identify interacting protein partners of NTL6,the SUCROSE NON-FERMENTING-1-RELATED PROTEIN KINASE 2.8(SnRK2.8)kinase was identified(Kim et al.2012;Umezawa et al.2004).Concomitant with interaction between SnRK2.8 and NTL6,the NTL6 transcription factor is phosphorylated by SnRK2.8(Kim et al.2012).The Thr142 residue is critical for NTL6 phosphorylation,and substitution of this residue strongly impairs its phosphorylation.Notably,phosphorylation of NTL6 at Thr142 is crucial for its nuclear import.Nuclear localization of NTL6 is substantially reduced in snrk2.8-deficient mutants,and a similar result is obtained upon mutation of the Thr142 phosphorylation site(Kim et al.2012).

Processing of NTL6 is uninfluenced by its phosphorylation status.NTL6 protein harboring T142A substitution(mNTL6)is still proteolytically processed by ABA and cold stress.Thus,it seems likely that proteolytic processing is followed by NTL6 phosphorylation.Indeed,processed nuclear NTL6 form(6ΔC)can also interact with SnRK2.8 kinase in the cytoplasm(Kim et al.2012).Furthermore,nuclear import of 6ΔC is inhibited in snrk2.8-deficient mutants,demonstrating that NTL6 phosphorylation occurs after processing and is not associated with proteolytic processing(Figure 1).

Taken together,controlled intracellular movement adds a level of complexity to MTF regulation.Regulations at multiple steps,such as transcription,proteolytic processing,and intracellular movement,not only elaborate MTF action but also give rise to crosstalk with additional network elements,enabling integration of a variety of input signals into the signaling pathway mediated by MTFs.

CONCLUDING REMARKS

Membrane-bound transcription factors have emerged as a way to regulate transcription factor activity in response to changes in physical and chemical properties of membranes,which are influenced by environmental and developmental changes.Since biological membranes perceive internal physiological alteration and environmental fluctuations and reflect initial changes in cellular properties,it enables plants to respond promptly to internal and/or external cues.MTFs are widely conserved in both monocots and dicots,and are involved in a variety of developmental processes,underscoring their biological relevance as essential signal transmitters.

Despite the biological importance of MTFs,a number of questions remain.Information on proteolysis of MTFs,other than bZIP MTFs,is largely fragmented.The specific proteases and E3 ligases that are involved in this process and how the processing machinery recognizes and cleaves MTFs remain largely unknown.The mechanisms for intracellular movement of MTFs are also generalized based on limited observations from bZIP28 and NTL6 actions.To expand our knowledge of plant MTFs,it will be necessary to characterize additional regulatory proteins involved in the control of MTF activity and dissect signal transduction pathways mediated by MTFs in more detail.

Structural analysis of MTFs should contribute significantly towards addressing fundamental questions in this field.It is hypothesized that physicochemical properties of membranes determine the 3D structure of MTFs,and that alteration of the protein structure of MTFs increases their accessibility and susceptibility to proteases or ubiquitination machineries.Therefore,they would be important research objectives to determine how internal and external stimuli modulate membrane structures and how MTFs are preferentially folded according to the composition of the surrounding membrane.

It has been estimated that approximately 10%of transcription factors are anchored in the membrane(Kim et al.2007b).In addition,recent bioinformatics approaches have shown that novel protein families,such as phospholipid scramblases and Tubby-like proteins,also have membrane-associated members(Bateman et al.2009),further supporting that many more MTFs are encoded in plant genomes.Accordingly,the repertoire of mode of action of MTFs and their regulatory mechanisms may be much more diverse.Further research will determine the physiological and molecular function of MTFs and provide concrete mechanistic insight into how signals perceived at the membrane are transmitted to the nucleus.

ACKNOWLEDGEMENTS

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education (NRF-2013R1A1A1004831)and by research funds of Chonbuk National University in 2012.

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