A new catch in the SNARE

A new catch in the SNARERejane Pratelli*, Jens-Uwe Sutter* and Michael R. Blatt

Laboratory of Plant Physiology and Biophysics, IBLS – Plant Sciences, Bower Building, University of Glasgow, Glasgow, UK G12 8QQ

Vesicle traffic underpins cell homeostasis, growth and

development in plants. Traffic is facilitated by a super-

family of proteins known as SNAREs (soluble N-ethyl-

maleimide-sensitive fusion protein attachment protein

receptors) that interact to draw vesicle and target mem-

brane surfaces together for fusion of the bilayers. Sev-

eral recent findings now indicate that plant SNAREs

might not be limited to the conventional ‘housekeep-

ing’ activities commonly attributed to vesicle traffick-

ing. In the past five years, six different SNAREs have

been implicated in stomatal movements, gravisensing

and pathogen resistance. These proteins almost cer-

tainly do contribute to specific membrane fusion events

but they are also essential for signal transduction and

response. Some SNAREs can modulate the activity of

non-SNARE proteins, notably ion channels. Other

examples might reflect SNARE interactions with differ-

ent scaffolding and structural components of the cell.

Eukaryotic cells maintain a range of membrane-delimitedcompartments that provide scaffolding to localize bio-chemical reactions, to confine proteins and their activitieswithin the cell, and to compartmentalize soluble com-pounds. To maintain this structural differentiation, thesecells use biosynthetic activities localized to the endoplas-mic reticulum (ER) and Golgi apparatus, and shuttlemembrane vesicles and their contents via membranefusion events between endomembrane compartments,the plasma membrane and the extracellular space. Vesicletraffic is crucial for nervous signal transmission across thesynaptic junctions of nerves and for cell wall delivery andbudding in yeast [1,2]. In plants, directed vesicle traffic isessential for maintaining cell polarity, growth and devel-opment, and is responsible for the compartmentationunderpinning the synthesis and delivery of some of themost biologically interesting and commercially importantproducts, including various alkaloids, anticancer drugs,dyes and enzymes [3,4]. These are highly dynamicprocesses involving the rapid turnover of large areas ofmembranesurface. Insomecell types,notably pollen,vesicletraffic can drive the turnover of plasma membrane surfacearea in excess of 0.01 cm2 min21 at the growing tip [3].

For such seamlessly rapid integration of transport, theplant cell must achieve two ends. First, it must overcomethe immense hydration force of the lipid bilayers in anaqueous environment [5]. Second, the cell must matchvesicles with their destination(s) to ensure efficienttargeting and delivery of specific membrane proteins and

soluble cargo. These two functions are carried out by asuperfamily of membrane and membrane-associatedproteins known as SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors).Subsets of SNAREs occur at vesicle and target membranesand interact to form a tetrameric bundle of coiled helices(Figure 1) that draws the membrane surfaces together andthus provide a mechanism for recognition, docking andfusion [1,2].

SNARE elements differ widely in size and structure butshare common structural (so-called SNARE) motifs thatcontribute to the protein–protein interactions at the coreof the SNARE complex. In mammalian tissues [1,6] and, inseveral instances, in plants [3], the canonical core of fourbundled a-helices of the SNARE complex are derived fromthree different membrane-anchored proteins (Figure 1),two on the target membrane (t-SNAREs) and one on thevesicle (v-SNARE). Variations on this theme include thesoluble yeast SNARE Vam7p [7] and the lipid-anchoredSNAREs AtYkt61 and AtYkt62 in Arabidopsis (Table 1)Table 2 [3]. However, in every case, functional SNAREcomplexes appear to comprise one element of each of foursubmotif domains, designated Qa, Qb, Qc and R [6]. The Qa,Qb and Qc domains centre about a glutamine residuewithin the SNARE motif of the t-SNAREs; the R domaincentres about an arginine residue of the complementarySNARE motif of the v-SNARE. This combinatorial modelfor the SNARE interactions goes some way to explainingboth their high specificity and, in some instances, theirredundancy [2]. It is also a key to understanding thediversity of SNAREs in Arabidopsis (see below) and,presumably, other plants. Both operational (v- and t-) andstructural (R- and Q-) designations are in common use; forconsistency, we use the structural designations here.

Mechanics of SNARE-driven fusion

In themselves, SNAREs are sufficient to drive fusionin vitro and, when expressed to expose the SNARE motifsoutside the cell, they facilitate fusion between mammaliancells [8]. Conversely, proteolytic cleavage of severalSNAREs by Clostridia botulinum neurotoxins blocksvesicle fusion and neurotransmitter release in vivo [9].Each toxin cleaves a unique protein target and thisspecificity has provided a powerful set of tools for probingSNARE function in vitro and in vivo in both animals[10,11] and plants [12,13]. These experiments providestrong evidence for SNARE function but do not rule out therole of lipids [14] and other (possibly regulatory) proteinsin fusion.

A mechanistic view of SNARE action in fusion is alsoconsistent with evidence that the linker between the

* These authors contributed equally to the publication.Corresponding author: Michael R. Blatt ([email protected]).

Review TRENDS in Plant Science Vol.9 No.4 April 2004

www.sciencedirect.com 1360-1385/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2004.02.007

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Figure 1. Mechanics of vesicle fusion and the SNARE cycle. (a) SNARE proteins at the vesicle (dark pink) and target (blue) membranes interact to bring the two membrane

surfaces together (1,2). Hemifusion follows with adjacent lipid monolayers conjoined (3) either by rearrangement of phospholipid acyl chains [57] or by insertion of hydro-

phobic molecules into intramembraneous areas [58]. The hemifusion state is thought to be stabilized to give a ‘readily releasable pool’ of exocytotic vesicles in some mam-

malian tissues [1,2]. Final fusion (and release of the vesicle contents as indicated) occurs with the merging of the two trans-lipid layers into a continuous fusion pore (4–7).

Kiss-and-run release of vesicle contents without intercalation of the membrane faces arises from tethering and a short-lived fusion (8) before the bilayers disengage (9,10).

(b) The SNARE core complex comprises a supercoil of four a-helical coils. The SNARE motifs of these coils are designated Qa, Qb, Qc and R, and often derive from three pro-

teins – two Q-SNAREs (e.g. syntaxin and SNAP25; also known as target or t-SNAREs) and one R-SNARE (e.g. VAMP/Synaptobrevin; also known as a vesicle or v-SNARE).

R- and Qa-SNAREs are colour-coded as in (a) above. The four a-helical coils interact by ‘zipping’ together to create a stable hydrophobic core centred about a cross-sectional

(so-called ‘zero’) layer of interaction between the three glutamine and one arginine residues. Syntaxin Q-SNAREs include a set of regulatory a-helices (Ha–Hb–Hc) that, in

the closed conformation, fold back to interact with the SNARE motif (or H3, as shown comprising a Qa motif) coil [2,3]. Both Q- and R-SNAREs can include transmembrane

domains (TM) or can be covalently anchored to lipid at conserved cysteines (CC). (c) Regulation of the SNARE cycle. SNARE components as indicated: dark pink, R-SNARE;

blue, syntaxin-like Qa-SNARE; yellow, Qb- and Qc-SNAREs. The cycle is initiated with release of the closed conformation by Sec protein binding to the Ha–Hb–Hc domain

to expose the syntaxin H3 coil. Association in the trans complex is accompanied by a large increase in core a-helical structure that helps to drive transition to the cis com-

plex [1]. Dissociation of the cis-complex and repriming requires binding of a-SNAP and the NSF ATPase, and ATP hydrolysis. Figure redrawn with elements from Ref. [1].

TRENDS in Plant Science

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5

6

7

8

9

10

(a)

Core complexR Qa

Qb

Qc

CC

(v-) R-SNARE (t-) Q-SNAREs

Sec

NSFα-SNAP

Loose trans-complex

Tight trans-complex

cis-complex

cis-complex

QbQc

RQa

(b)

(c)

QaQb Qc Ha Hb HcTM TMR

SNAP25 VAMP Syntaxin

Open Closed


www.sciencedirect.com


Table 1. Arabidopsis Q-SNAREs, related proteins and their partners

Namea Synonymsb AGI designationc Localizationd Partner interactionse

Qa SNARE, syntaxin-like

Subclass Syp1

AtSyp111 Knolle At1g08350 Phragmoplast AtSnp11,

AtSnp12, AtSnp13

AtSyp112 At2g18260

AtSyp121 AtSyr1, Pen1 At3g11820 Plasma membrane

AtSyp122 AtSyr4 At3g52400 Plasma membrane AtSnp11

AtSyp123 At4g03330

AtSyp124 At1g55410

AtSyp125 At1g10980

AtSyp131 At3g03800

AtSyp132 At5g08080

Subclass Syp2

AtSyp21 AtPep12 At5g16830 Prevacuole AtVti11, AtSyp51

AtSyp22 AtVam3 At5g46860 (Pre)vacuole

AtSyp23 AtPlp At4g17730

Subclass Syp3

AtSyp31 AtSed5 At5g05760

AtSyp32 At3g24350

Subclass Syp4

AtSyp41 AtTlg2a At5g26980 trans-Golgi AtSyp61, AtVps45

AtSyp42 AtTlg2b At4g02195 trans-Golgi AtSyp61, AtVps45

AtSyp43 At3g05710

Subclass Syp8

AtSyp81 At1g47920

Qc SNARE, syntaxin-like

Subclass Syp5

AtSyp51 At1g15930 trans-Golgi network, prevacuole AtSyp61, AtSyp21

AtSyp52 At1g73260

Subclass Syp6

AtSyp61 Osm1 At1g27550 trans-Golgi network, AtSyp41, AtSyp51

prevacuole

Subclass Syp7

AtSyp71 At3g09740

AtSyp72 At3g45280

AtSyp73 At3g61450

Qb SNARE, Bet1-like

AtBet11 BS14a At3g58170

AtBet12 BS14b At4g14450

Qb SNARE, Gos1-like

AtGos11 At1g15590

AtGos12 Atg245200

Qb SNARE, Membrin-like

AtMemb11 At2g36900

AtMemb12 At5g50440

Qb SNARE, Plant specific

AtNpsn11 At2g35190

AtNpsn12 At1g44640

AtNpsn13 At3g17440

Qb SNARE, Vti1-like

AtVti11 AtVTI1a At5g38510 trans-Golgi network, AtSyp21, AtSyp51

prevacuole

AtVti12 AtVTI1b At1g25740 trans-Golgi network, AtSyp61, AtSyp51

prevacuole

AtVti13 At3g29100

Qb1c SNARE, SNAP25-like

AtSnp11 AtSNAP33 At5g61210 Plasma membrane, AtSyp111, AtSyp122

phragmoplast

AtSnp12 AtSNAP29 At5g07880 AtSyp111

AtSnp13 AtSNAP30 At1g13530 AtSyp111

aFor naming conventions, see Refs [3,22].bSynonyms, original or previously used names (see text and Figure 2 for references).cArabidopsis Genome Initiative designation.dSee text and Figure 2 for details; also Ref. [3]. Line left blank if not known.eKnown partner interactions either in vitro or in vivo (see text for details, also Ref. [3]). Line left blank if not known.

Review TRENDS in Plant Science Vol.9 No.4 April 2004 189



SNARE motif and membrane anchor determines thecharacteristics of fusion. Both the amino acid compositionand the length of this region affect the kinetics of fusion[15] and, indeed, might explain different kinetic modesidentified in fusion events in both plants [3] and animals[16]. One mode, in which the SNARE complex does notassemble fully, leads to patterns of ‘kiss-and-run’ fusionwith vesicles releasing their cargo but not fully integratingwith the target membrane (Figure 1). Conceivably,interactions between one or more SNARE elements –Q- and/or R-SNAREs – with shorter (and presumablyless flexible) linker(s) might favour rapid and complete

fusion, whereas interactions with other SNARE partnersincorporating longer (more flexible) linker(s) could driveslow or incomplete (kiss-and-run) fusion. Other combi-nations could lead to a SNARE complex that is stable butrequires an additional protein(s) to complete the transitionfrom hemifusion to complete integration with the targetmembrane. In short, the potential for complex formationwith different partners might be one key factor in thetargeting and kinetic flexibility of intracellular traffic.

The assembled SNARE complex shares considerablestructural homology with viral type-I fusion proteins[1,17]. The most intensively studied members of this

Table 2. Arabidopsis R-SNAREs, SNARE-related proteins and their partners

Namea Synonymb AGI designationc Localizationd Partner interactionse

R SNARE, VAMP-like

AtVamp711 VAMP7C At4g32150

AtVamp712 At2g25340

AtVamp713 At5g11150

AtVamp714 At5g22360

AtVamp721 Sar1 At1g04630

AtVamp722 VAMP7B At2g33120

AtVamp723 At2g33110

AtVamp724 At4g15780

AtVamp725 At2g32670

AtVamp726 At1g04650

AtVamp727 At3g54300

AtSec22 At1g11610

AtYkt61 At5g58060

AtYkt62 At5g58180

R SNARE, Tomosyn-like

AtTyn11 At5g05570

AtTyn12 At4g35560

SNARE-associated elements

aSNAP-like

AtAsnp11 a-SNAP1 At3g56450

AtAsnp12 a-SNAP2 At3g56190

AtAsnp21 g-SNAP At4g20410

ATPases

AtNSF At4g04910

AtCDC48a At3g09840

AtCDC48b At2g03670

AtCDC48c At3g01610

(Sec1-like)

AtSec11 Keule At1g12080 Phragmoplast

AtSec21 AtSEC1a At1g01980

AtSec22 AtSEC1b At4g12120

Vps- and Sly-like

AtVps45 At1g70890 trans-Golgi network AtSyp41,AtSyp42

AtVps33 At3g54860

AtSly1 At2g17980

VAP33-like

AtPva11 At3g60600 Endoplasmic reticulum

AtPva12 At2g45140

AtPva13 At4g00170

AtPva14 At1g51270

AtPva21 At5g47180

AtPva22 At1g08820

AtPva31 At2g23830

AtPva41 At5g54110 Plasma membrane

AtPva42 At4g21450

AtPva43 At4g05060

aFor naming conventions, see Refs [3,22].bSynonyms, original or previously used names (see text and Figure 2 for references).cArabidopsis Genome Initiative designation.dSee text and Figure 2 for details; also [3]. Line left blank if not known.eKnown partner interactions either in vitro or in vivo (see text for details, also Ref. [3]). Line left blank if not known.




group are influenza haemagglutinin protein and gp41 ofHIV. Both are single membrane proteins that anchor thevirus, tethering viral and host membranes, and thenundergo a conformational change that brings the twomembranes into close apposition and drives fusion. It hasbeen suggested that the peptide sequence inserted in thehost membrane both anchors and deforms the bilayer tofavour transition to a hemifusion structure and hence toforce fusion [18]. There are similarities, too, in thestoichiometry of protein (complex) units between the twofusion processes. Fusion driven by haemagglutinin iscooperative, engaging two or three fusion initials to openthe fusion pore [19], whereas measurements of intracellu-lar calcium stoichiometry indicate that three to fourSNARE complexes (one Ca2þ ion per complex) co-operatein Ca2þ-dependent exocytosis [20]. Conceivably, then, thenumber of SNARE complexes required might depend onthe working distance and deformational force that a singlecomplex can apply between two membranes. If so, fewerSNARE complexes might be required per fusing vesiclewhen the complexes incorporate shorter linkers betweenthe membrane anchors and SNARE motifs. An interestingtest of this hypothesis would entail measuring the calciumdependency of single exocytotic events to see if the SNARE(Ca2þ:fusion-event) stoichiometry could be affected bymodifying the linker region of the individual SNAREproteins.

SNARE genomics

The availability of the complete genome sequence ofseveral model eukaryotes, including Arabidopsis [21],has provided considerable information about vesicletrafficking elements, not only their overall conservationbut also the remarkable degree of complexity within eachsubfamily of these proteins. Comparisons with animal(Homo sapiens, Caenorhabditis elegans), insect (Droso-phila) and yeast (Saccharomyces cerevisiae) models turnup several important distinctions, including a diversity ofSNARE proteins (68 in Arabidopsis compared with 35 inhumans, 21 in yeast and 20 in Drosophila) as well asseveral unique gene subfamilies encoding proteins withwholly unknown function(s) in Arabidopsis. It is likelythat new functions underlie this diversity of proteins, butassigning biological roles is difficult, even where closehomologies do occur across phylogenetic boundaries.Individual SNARE proteins can perform different func-tions at different sites within the cell and, in someinstances, can even swap functions between cognateinteractors [22].

The core of 68 putative vesicle trafficking proteins(Table 1) in the Arabidopsis genome, predicted on the basisof sequence domain homology, are all expressed.These proteins include 24 syntaxin-like Q-SNAREs,three SNAP25 Q-SNARE homologues and 14 VAMP-like R-SNAREs [3]. The Arabidopsis genome also encodes


Cell wall

Anterograde

Retrograde

Exocytosis

Endocytosis

Endoplasmic reticulum

Golgi

Vacuole

Nucleus

Syp81

Syp42Syp43(Tlg2)

Syp41

Syp31Syp32(Sed5)

Syp131Syp132

Syp51Syp52(Tlg1)

Syp111(Knolle)Syp112

Syp121Syp122Syp123Syp124Syp125

Syp61

Syp21(Pep12)Syp22(Vam3)Syp23

Plasma membrane

Syp71Syp72Syp73

Figure 2. Functional localization of Arabidopsis syntaxin-like Q-SNAREs, known (blue text) or presumed (black), based on homologies with mammal and yeast SNAREs.

Arabidopsis syntaxin-like Q-SNAREs are distributed between the major vesicle-trafficking pathways of the cell. Syp1 proteins show closest homology to the plasma mem-

brane syntaxins Sso1/Sso2 of yeast and humans and, of these, AtSyp121 and AtSyp122 are known to be localized to the plasma membrane [12,48]. AtSyp111 is found

exclusively at the phragmoplast of dividing cells [34]. Syp3 and Syp8 proteins show the greatest homology to Golgi and endoplasmic reticulum syntaxins Sed5p, Ufe1p of

yeast and human Syn18 [59,60]. The Syp2 and Syp4 proteins include orthologues found in yeast at the trans-Golgi network (TGN) and pre-vacuolar compartment (PVC),

and localize similarly [3,23]. The Syp5 and Syp6 classes show homology to Tlg1p and Tlg2p of yeast, and to human Syn6 and Syn8, which contribute to endosomal traffick-

ing [2]. Of these, AtSyp51 and AtSyp61 are associated with the PVC and the TGN, respectively [30]. The Syp7 class shows no clear homology to other eukaryotic SNAREs

and the function(s) of these proteins is still unknown.




three SNAP homologues, one NSF ATPase, six Sec1-likeperipheral binding proteins (which regulate syntaxinaccessibility and SNARE interactions in mammals, yeastand Drosophila [1,2]) and a selection of over 30 otherassociated proteins that might have functions in scaffold-ing and cytoskeletal interactions.

As a family, the best characterized to date are thesyntaxin-like Q-SNAREs. These proteins divide betweenfour groups based on sequence homologies that alignbroadly with their anticipated subcellular locations andfunctions (Table 1, Figure 2) [3], and further subdivide intoeight classes (the syntaxins of plants, Syp1–Syp8) basedon genomic analyses [23]. All show the hallmarks ofsyntaxins (Figure 1), notably three N-terminal coildomains (Ha, Hb, Hc), the H3 coil and C-terminaltransmembrane domains. In spite of the multiplicity ofproteins within these classes, surprisingly little directevidence of functional redundancy has yet come to light.Gene disruptions and mutations of single members inthree of the Syp classes lead to lethal phenotypes, althoughat least one additional gene occurs within each class[3]. T-DNA knockouts of AtSyp121 and AtSyp122 donot show obvious differences from the wild type in theglasshouse (F. Assaad, pers. commun.) but mutation ordeletion of AtSyp121 does affect pathogen resistance(see below). Some specialization relates to spatial, tem-poral or developmental targeting, as is the case forAtSyp112, which is expressed in leaves and flowers butcomplements the embryo-lethal knolle (Atsyp111) mutantof Arabidopsis when targeted to the cell plate [24]. Bycontrast, the plasma membrane Q-SNARE AtSyp121(AtSyr1) fails to rescue the knolle mutant [24], evenwhen targeted to the cell plate. Danny Geelen et al. [25]found that expression of a dominant-negative mutant ofthe tobacco orthologue NtSyp121 (NtSyr1) did not increasethe number of dividing initials or give rise to multinucleatecells ‘trapped’ late in division. The absence of suchtrapping, a characteristic of the knolle mutant, also arguesagainst a functional overlap between these two syntaxins.Of the Arabidopsis SNAP25 Q-SNARE orthologues,Maren Heese et al. [26] have reported that AtSnp11(AtSNAP33) interacts with AtSyp111 in yeast two-hybridscreens, accumulates at the plasma membrane and,during cell division, localizes with AtSyp111 at thephragmoplast. Knockout of AtSnp11 resulted in a dwarfphenotype that was eventually lethal. Because AtSyp111also interacts with the two other SNAP25-like proteinsin vitro, AtSnp11 function might be subsumed in part bythe other SNAP25 homologues.

Much less is known of the vesicle-associated proteins, orR-SNAREs, in Arabidopsis. The Arabidopsis VAMP iso-forms show greatest homology to the mammalian VAMP7proteins that contribute to endosomal and lysosomaltrafficking [2], Sec22 [27] and the lipid-anchored yeastR-SNARE Ykt6p [28]. Notable by their absence arehomologues to VAMP1 associated with secretory traffic tothe plasma membrane in neuromuscular tissues [2]. TheVti1p-like R-SNARE homologues AtVti11 (AtVti1a) andAtVti12 (AtVTI1b) do appear to parallel elements of theyeast trans-Golgi-network (TGN)-to-vacuole traffickingpathways [29,30] in associating with the vacuolar markers

AtSyp21, AtSyp51 and AtSyp61, and in suppressingthe vti1p-mutant phenotype in yeast. Arabidopsis alsoharbours two genes with sequence similarity to theGolgi-associated Bet1p/Sft1p subfamilies of yeast andrescue growth in temperature-sensitive sft1-1 and Dsft1mutant yeast [31].

Control of the interactions between cognate SNAREelements is of paramount importance to the cell, if onlybecause many of these proteins are synthesized at the ERand pass through the Golgi before reaching their site(s) ofaction. Several families of putative binding proteinsknown to associate with SNAREs are found in theArabidopsis genome [3]. Of these, homologues of theyeast and mammalian Sec1 family of proteins probablyaffect the stability of the so-called ‘closed’ conformation ofthe syntaxin (Figure 1) that suppresses promiscuousSNARE coupling [2]. Arabidopsis AtSec11 (Keule;Table 1) shows the closest homology to neuronal nSec1and the yeast Sec1p. AtSec11 is required for cytokinesisand was isolated in mutant screens along with AtSyp111,with which it binds and colocalizes [26,32]. One otherperipheral binding protein examined to date, AtVps45, ispresent on the TGN and interacts with two Q-SNAREs,AtSyp41 and AtSyp42, that show similarities to the yeastQ-SNARE Tlg2p [33].

Do plant SNAREs function in vesicle trafficking?Almost certainly. However, there is still little directevidence to support specific functions of these proteins.To date, only Geelen et al. [25] have provided evidence for atrafficking function in vivo, demonstrating that thecytosolic domain of the tobacco Q-SNARE NtSyp121 actsto suppress traffic of a secreted green fluorescent protein(secGFP) marker. Expression of the NtSyp121 cytosolicdomain led to an accumulation of secGFP in the ER andGolgi, and a cessation of growth that could be rescued byoverexpression of the full-length NtSyp121 protein. Theseobservations are most easily understood if binding of thecytosolic domain forms non-functional SNARE complexesand thereby suppresses vesicle traffic to the plasmamembrane. Other evidence includes phenotypic analyses,notably the accumulation of vesicular structures aroundthe cell plate in Arabidopsis AtSyp111 (knolle) mutantsthat are arrested late in cell division [34,35].

SNAREs in signalling

Several recent findings indicate that SNARE functions inplants are not limited to a role in vesicle trafficking alone.Genetic and functional screens unrelated to traffickinghave uncovered SNARE proteins in evoked responses asdiverse as stomata movements, gravitropism and patho-gen resistance. Some of these observations might comp-lement findings of SNAREs and their partners inmammalian tissues that regulate ion transport associatedwith signalling and adaptation, either by direct inter-actions, for example with ion channels [36–39], bycontrolling transporter protein density in target mem-branes [40], or through interactions with membranereceptors [41]. So, it will be interesting to see whethercomplementary and/or additional functions come to lightin plants.




Abscisic acid and water stress

The first indication of other roles for SNAREs came withthe identification in our laboratory of a syntaxin-likeprotein NtSyp121 (NtSyr1) from tobacco using anexpression-cloning strategy in a hunt for an abscisic acid(ABA) receptor [12]. NtSyp121 was observed throughoutthe plant, localized to the plasma membrane [42], andintroducing a soluble, truncated (dominant-negative) formof the protein was found to block traffic to the plasmamembrane in vivo [25]. Intriguingly, the expression ofNtSyp121 was strongly and transiently induced in tobaccoleaves (but not in roots) by ABA, drought, salt andwounding [12,42]. Furthermore, the same truncatedform of NtSyp121, as well as treatment with BotN/C(which cleaved the NtSyp121 protein), prevented ABAaction in modulating Kþ and Cl2 channels in guard cells[12]. Channel modulation, which is a prerequisite forstomatal closure in ABA, normally takes place within1–2 min and depends on signal cascades coupled tochanges in cytosolic free Ca2þ concentration and pH[43,44]. Thus, in addition to a ‘housekeeping’ function invesicle trafficking that might be related to the changes incell surface area during stomatal movements, the tobaccoQ-SNARE seems to play a role in the leaves, possiblythrough direct interaction with ion channels (Figure 3)that transduce ABA, wounding and related stress signals.

It is noteworthy that some of the phenotypic charac-teristics associated with expression of the dominant-negative NtSyp121 are similar to those in mutants ofanother Q-SNARE, AtSyp61 of Arabidopsis. Jiang-KangZhu et al. [45] isolated the osm1 (Atsyp61) mutant in ascreen for salt-sensitive growth and found that it affectedstomatal movement as well as root morphology. AtSyp61 is

thought to play a role in vesicle traffic to the pre-vacuolarcompartment [30] and its disruption might therefore affectosmotic solute balance in the vacuole and/or the regulationof transport activities at these membranes.

Pathogen resistance

Two recent genetic screens for fungal pathogen resistancein Arabidopsis and barley have yielded Q-SNAREsorthologous to NtSyp121. Nicholas Collins et al. [46]identified mutations of AtSyp121 (PEN1) and of HvSyp121(ROR2) that facilitate non-host penetration of the leafsurface by germinating hyphae of powdery mildew.Although AtSyp121 expression in Arabidopsis is affectedby ABA [12], the pen1-1 null mutant did not show anydefects in plant growth, stomatal behaviour or ABAregulation of seed germination and root development.Thus, in contrast to NtSyp121, AtSyp121 action inresistance to fungal penetration appears to be independentof ABA. Wild-type AtSyp121 and HvSyp121 localized tothe plasma membrane and, in infected leaves of Hvsyp121-mutant barley, large aggregates of vesicles containingreactive oxygen species (ROS) were often seen to accumu-late within the cells.

Because ROS are involved in cell-wall cross-linkingand signalling functions during pathogen attack,Collins et al. [46] suggested that a failure of thesevesicles to fuse with the plasma membrane couldimpair resistance to fungal penetration by preventingthe release of toxic compounds and callose secretion.Nonetheless, the observations do not rule out otherroles for the Q-SNAREs, including direct interactionswith Ca2þ channels (Figure 3) to affect Ca2þ signallingthat contributes to wounding and pathogen responses

Figure 3. Five generic modes for SNARE integration in cellular signalling events. The local event sequence flows from left to right. Vesicle trafficking is capable of delivery

(1) and SNAREs (dark pink, R-SNARE; blue, Q-SNARE) can be important for the selective removal (2) of different ion channels (two ion channel species, here shown as

green and purple) and other proteins from the target membrane [40], here identified as the plasma membrane. Direct interactions with SNAREs affect the activity of several

ion channels (3), including Ca2þ channels [36], Cl2 channels [37] and Kþ channels [39]. Direct interactions with SNAREs also modulate ion channel response to other signals

(4), such as activation by heterotrimeric G-proteins [11,38]. Finally, SNAREs and associated proteins can contribute to cytoskeletal organization (5) and provide a link to sub-

membrane scaffolding networks [55,61].


Plasma membrane

vesicle

4

In

Out

2

53

1

Closed and open channel CytoskeletonClosed and open Q-SNARE Second messenger component

R-SNARE Cytosolic scaffolding element




in plants [47]. Intriguingly, the closely relatedQ-SNARE AtSyp122 is phosphorylated in Arabidopsiscell cultures on elicitation by bacterial flagellin [48].Whether direct participants or downstream targets insignalling, the fact that two plasma membrane Q-SNAREsin Arabidopsis are associated with pathogenicity high-lights the importance of these proteins in the cellularstimulus–response coupling of defence.

Gravitropism

AtSyp22 and AtVti11 are members of the same SNAREcomplex in Arabidopsis that are thought to associate withvesicle traffic between the TGN, pre-vacuolar compart-ment and vacuole (Table 1, Figure 2). Remarkably,mutants of both proteins suppress shoot gravitropism,which results in altered stem morphology [49,50]. At thecellular level, the mutants correlate with an abnormaldistribution of amyloplasts and vacuolar fragmentation.The single amino acid substitution of the Atsyp22 mutantreduced SNARE complex stability in vitro, leadingDaisuke Yano et al. [50] to speculate that it might affectvacuole ‘plasticity’ in a way that interferes with the freesedimentation of the amyloplasts within the cell and hencewith perception of gravitational direction.

Another tempting hypothesis is that these mutationsaffect connections between the SNARE complex andscaffolding elements to alter cytoskeletal superstructureand affect movement of the amyloplasts or their inter-action(s) with plasma membrane or vacuolar membraneproteins (Figure 3). This interpretation finds support withincreasing evidence for various scaffolding proteins invesicle tethering of yeast, some of which interact with thecytoskeleton and are often recruited by members of theRab and Arf GTPase families, that also contribute toSNARE complex formation [51,52]. Indeed, cell-surfacedistribution of the auxin efflux carriers that contribute topolar and tropic growth in Arabidopsis are remarkablysensitive to factors that affect vesicle traffic and cyto-skeletal organization [53,54]. It accords, too, with a role forvesicle traffic in determining cytoskeletal distribution [55]and for the actin cytoskeleton as a key determinant inamyloplast movement and gravistimulus transmission [56].

Conclusion

Research over the past five years has shown that SNAREproteins in plants have roles in cellular stimulus–responsecoupling that lie well outside the boundaries of theconventional ‘housekeeping’ functions commonly attributedto vesicle trafficking. Although most of these proteinsprobably do contribute to specific membrane fusion events,some could also be important components of signallingpathways. These unexpected functions might relate to theability of some SNAREs to modulate the activity of non-SNARE proteins (such as ion channels) or to interact withstructural components of the cell. It will now be necessary toadd molecular details of these potential SNARE proteinpartners and to identify the signalling networks in whichtheyparticipate.Futureworkmustalsoaddressquestionsofmechanistic overlap between stimulus–response couplingand vesicle trafficking, notably in cargo and selectivemembrane protein cycling within the cell.

AcknowledgementsThis work was supported by BBSRC grants P12750, C13599, C09640 andP13610 to M.R.B.

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