Ca2+–synaptotagmin directly regulates t-SNARE functionduring reconstituted membrane fusionAkhil Bhalla1–4, Michael C Chicka1,2,4, Ward C Tucker2 & Edwin R Chapman1,2

In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca2+ and synaptotagmin-1 (syt). Ca2+ promotes theinteraction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we haveused a defined reconstituted fusion assay to determine directly whether syt–t-SNARE interactions couple Ca2+ to membrane fusionby comparing the effects of Ca2+–syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p)SNAREs. Ca2+–syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids.However, Ca2+–syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs.Thus, Ca2+–syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca2+–sytdrove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca2+–syt alters t-SNARE structure.

Intracellular membrane fusion is mediated by the action of soluble NSF(N-ethylmaleimide-sensitive factor)-attachment receptor (SNARE)proteins on vesicle (v-SNARE) and target (t-SNARE) membranes1.In nerve terminals, Ca2+-triggered exocytosis of neurotransmittersrequires the v-SNARE synaptobrevin (syb; also called VAMP) andthe t-SNAREs syntaxin and SNAP-25 (refs. 2,3). The cytoplasmicdomains of syb, syntaxin and SNAP-25 assemble into parallelfour-helix bundles4–6. Syntaxin and syb contribute one helix each,and SNAP-25 contributes two helices4. The pairing of v- andt-SNAREs into trans SNARE complexes and the formation of helicalbundles have been proposed to provide the driving force for fusion4,7.In support of this idea, purified, reconstituted SNAREs have beenshown to mediate membrane fusion in vitro8,9.

Although it is well established that SNAREs have key roles inmembrane fusion1,3, the relationship between SNARE assembly andfusion remains unknown. In some reports, it has been suggested thatSNARE complex assembly proceeds in an N- to C-terminal direction(membrane distal to membrane proximal)10–12, whereas others haveproposed a concerted ‘one-step’ assembly mechanism13. It is likely thatassembly is tightly regulated, because purified reconstituted cognatev- and t-SNAREs form trans SNARE complexes that catalyze mem-brane fusion in a Ca2+-independent manner8,14. Therefore, duringCa2+-regulated fusion events, including synaptic vesicle exocytosis atnerve terminals, SNAREs must be regulated, either directly or indir-ectly, by Ca2+-binding proteins. Studies using permeabilized PC12cells support the idea that Ca2+ might be required for SNARE complexassembly. Namely, removal of one helix of SNAP-25 by botulinumneurotoxin E blocks secretion; exocytosis can be rescued by addingback fragments of SNAP-25, but these fragments must be added in thepresence of Ca2+ to restore secretion15. These data are consistent with

the idea that SNAP-25 assembles into SNARE complexes in a Ca2+-promoted manner.

Recent studies have provided strong support for the idea thatsynaptotagmins serve as Ca2+ sensors that regulate exocytosis16,17.Sixteen isoforms of synaptotagmin have been identified; syt is thebest-characterized form and is localized to synaptic and large densecore vesicles18–20. Biochemical studies have revealed that syt formsspecific21,22, stoichiometric23–25 Ca2+-promoted complexes with thet-SNAREs syntaxin and SNAP-25. Syt also binds with highaffinity to anionic phospholipids (such as phosphatidylserine(PS))26,27. The interactions of syt with t-SNAREs and mem-branes are mediated by the tandem C2 domains of syt, designatedC2A and C2B17.

Whether t-SNAREs or anionic lipids are essential effectors of Ca2+–syt action is a crucial issue that has yet to be resolved. A mutation thatimpairs C2A-PS interactions results in diminished exocytosis inhippocampal synapses28 and shifts the Ca2+ requirement for exo-cytosis in chromaffin cells29, but this mutation has more recently beenshown also to inhibit the binding of syt to SNAP-25 and to diminishC2B-PS interactions30. The recent reconstitution of Ca2+-regulatedmembrane fusion provides an alternative means to more directlyaddress these questions. In this reduced system, Ca2+ and syt regulateSNARE-mediated membrane fusion31. Omission of PS has no effecton SNARE-catalyzed fusion but completely abrogates stimulation offusion by Ca2+–syt32. These data provide direct support for the ideathat PS is a crucial effector for syt.

The goal of the current study was to use the reconstituted fusionassay to determine directly whether t-SNAREs are also essentialeffectors of Ca2+–syt. Using reconstituted vesicles harboring neuronalor yeast SNAREs, we demonstrate that syt–t-SNARE interactions are

Received 3 November 2005; accepted 21 February 2006; published online 26 March 2006; doi:10.1038/nsmb1076

1Howard Hughes Medical Institute, 2Department of Physiology and 3Molecular and Cellular Pharmacology Program, University of Wisconsin, 1300 University Avenue,SMI 129, Madison, Wisconsin, USA. 4These authors contributed equally to this work. Correspondence should be addressed to E.R.C. (chapman@physiology.wisc.edu).

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required for Ca2+–syt stimulation of SNARE-mediated membranefusion. We also show that Ca2+–syt drives the assembly of t-SNAREheterodimers in vitro and regulates a second step after t-SNAREcomplex assembly to facilitate mixing of the inner and outer leafletsof SNARE-bearing liposomes.

RESULTSCa2+–syt binds neuronal but not yeast t-SNAREsCa2+–syt facilitates SNARE-catalyzed membrane fusion in vitro31, butthe mechanism of stimulation is unclear. In this reduced assay system,Ca2+–syt interacts with the t-SNAREs syntaxin and SNAP-25(refs. 21,24,25) at all stages of SNARE complex assembly23,33. It hasalso been demonstrated that Ca2+–syt binds, with high affinity, toanionic phospholipids, including the PS present in the reconstitutedv- and t-SNARE vesicles26,31,34. Finally, in the presence of PS, Ca2+–sytassembles into homo-oligomeric structures35. Therefore, PS, t-SNAREsand other copies of syt are the only possible effectors for the action ofCa2+–syt in the fusion assay.

The role of PS in the reconstituted fusion assay has recently beenaddressed by omitting this phospholipid from the SNARE-bearingreconstituted vesicles. Omission of PS has no effect on SNARE-mediated membrane fusion, but it completely abolishes the abilityof Ca2+–syt to stimulate fusion, demonstrating that PS is an essentialeffector of Ca2+–syt action32. Here, we sought to carry out similarexperiments to address the function of Ca2+–syt–t-SNARE interac-tions by replacing neuronal SNAREs with SNARE proteins that Ca2+–syt is unable to bind. We reasoned that, as yeast lack syt, we might beable to use yeast SNARE proteins for these studies. We focused on aSNARE complex, Sso1p–Sec9c–Snc2p, that mediates exocytosis inyeast3,14. Sso1p, Sec9c and Snc2p are orthologs of syntaxin, SNAP-25 and synaptobrevin, respectively.

The ability of the cytoplasmic domain of syt to interact withneuronal and yeast SNAREs was assessed using a flotation assay(Fig. 1a)31,32. The flotation assay measures binding of syt to full-length SNAREs embedded in membranes rather than to solublefragments of SNAREs or SNAREs in detergent micelles31. TheSNARE-harboring vesicles used in these assays were composed of100% phosphatidylcholine (PC), a phospholipid that syt does notinteract with; hence, coflotation of syt is mediated by syt–SNAREinteractions rather than syt–membrane interactions. As reportedpreviously, syt interacted with neuronal t-SNARE heterodimers (syn-taxin and SNAP-25) in a Ca2+-promoted manner but did not bind theneuronal v-SNARE, syb (Fig. 1b, left)31,32. In control experiments,Ca2+–syt bound protein-free vesicles composed of 15% PS and 85%PC via its high affinity for PS (Fig. 1b, left and right)34. In contrast,Ca2+–syt did not bind the vesicles harboring yeast SNARE proteins,which included the yeast v-SNARE protein Snc2p, the yeast t-SNARE

protein Sso1p or the t-SNARE heterodimer composed of Sso1p andSec9c (Fig. 1b, right). These samples were also subjected to immuno-blotting analysis using an antibody to syt, which confirmed that therewas a Ca2+-stimulated interaction between syt and neuronalt-SNAREs, whereas little if any syt bound the yeast t-SNAREs(Fig. 1c). Longer exposures of the blot revealed a faint syt band inthe yeast v- and t-SNARE lanes, indicating that some degree ofnonspecific binding occurs (data not shown). These data show thatsyt specifically binds neuronal t-SNAREs in response to Ca2+ but doesnot bind yeast t-SNAREs.

Ca2+–syt stimulates fusion via interactions with SNAREsTo address the functional relevance of Ca2+–syt–t-SNARE inter-actions, we compared the ability of Ca2+–syt to enhance fusioncatalyzed by neuronal or yeast SNAREs in a reconstituted mem-brane fusion assay8,31. In this assay, fluorescence resonance energytransfer (FRET) donor (7-nitrobenz-2-oxa-1,3-diazole, or NBD) andacceptor (rhodamine) pairs, attached to the headgroup of phospho-lipids, are incorporated into the v-SNARE vesicles. Fusion ofthese labeled vesicles with unlabeled t-SNARE vesicles results indilution of the donor and acceptor, leading to an increase in thefluorescence of the donor (that is, NBD). This increase was convertedto rounds of fusion as described in Methods and SupplementaryMethods online.

As previously demonstrated, Ca2+–syt stimulated fusion betweenneuronal v- and t-SNARE vesicles (Fig. 2a, left)31. In marked contrast,Ca2+–syt did not stimulate fusion between yeast v- and t-SNAREvesicles (Fig. 2a, right), despite the fact that Ca2+–syt aggregatedneuronal and yeast SNARE-bearing vesicles to similar extents(Fig. 2b). The aggregation is largely due to Ca2+–syt–PS interactions,with only a minimal contribution from Ca2+–syt–t-SNARE inter-actions (Supplementary Fig. 1 online). From these data, we concludethat Ca2+–syt does not regulate SNARE-mediated fusion solelyby aggregating vesicles to drive apposition of v- and t-SNAREs.Rather, Ca2+–syt–t-SNARE interactions are crucial for regulatedmembrane fusion.

We note that the extent of fusion at 120 min was similar forneuronal and yeast SNAREs (Fig. 2a) and that the vesicles used inthese experiments harbored similar densities of SNARE proteins(Fig. 2a, insets). Therefore, it is unlikely that Ca2+–syt was withouteffect on yeast-mediated fusion owing to a lack of dynamic range inthe fusion assay. To address this question more thoroughly, we titratedSec9c (the yeast SNAP-25 homolog36) and syt in the fusion assay.Ca2+–syt was without effect at all concentrations of Sec9c tested, evenwhen the extent of fusion was low (Fig. 2c). In control experimentsusing neuronal SNAREs, increasing the concentration of syt in thepresence of Ca2+ greatly enhanced membrane fusion (Fig. 2d)31,32.

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Figure 1 Ca2+–syt binds neuronal, but not yeast, t-SNAREs. (a) Diagram of the flotation assay used to monitor binding of the cytoplasmic domain of syt to

reconstituted vesicles. Syt will cofloat with the vesicles only if it interacts with a constituent of the vesicles. For all reactions, one-third of each sample was

separated by SDS-PAGE and stained with Coomassie blue. (b) Syt binds neuronal t-SNAREs but not yeast t-SNAREs in response to Ca2+. Syt was incubated

with vesicles (100% PC) harboring either neuronal or yeast v- or t-SNAREs (Vr and Tr, respectively) or with PS-containing protein-free (Pf) vesicles in the

presence of EGTA (–) or Ca2+ (+). t-SNARE vesicles containing Sso1p alone (Sso1pr) or the Sso1p–Sec9c heterodimer (yeast Tr) were used to assay syt–yeast

t-SNARE interactions. (c) Indicated samples from b were analyzed by immunoblotting with an antibody to syt I.

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Thus, a potential effect of syt on yeast SNAREs was not masked by theexperimental setup; rather, Ca2+–syt is unable to stimulate fusionbetween yeast v- and t-SNARE vesicles.

Ca2+–syt–PS regulates interactions between SNARE proteinsThe data described above indicate that Ca2+–syt can directly ‘activate’t-SNAREs for membrane fusion. One possibility is that syt, in thepresence of Ca2+, aids in the assembly and folding of SNARE proteinsinto fusion-competent complexes. This idea is supported by previousstudies where catecholamine secretion from botulinum neurotoxin E(BoNT/E)-treated neuroendocrine cells was rescued by the recruit-ment of a SNAP-25 fragment only in the presence of Ca2+ (ref. 15).This suggests that SNARE assembly requires Ca2+ and, potentially, syt.This latter idea is further supported by the finding that mutations inDrosophila melanogaster syt can inhibit the formation of SDS-resistantSNARE complexes in vivo37.

We explored the possibility that Ca2+–syt regulates assembly ofSNARE proteins with one another by using t-SNARE vesicles thatcontained syntaxin alone rather than preformed syntaxin–SNAP-25heterodimers. It has previously been reported that SNAP-25 must beprebound to syntaxin, before reconstitution, to drive membranefusion8. We confirmed this by mixing the syntaxin-containing vesicleswith soluble SNAP-25 and v-SNARE vesicles in the presence orabsence of syt and Ca2+. The fusion data are shown in Figure 3a.In the absence of SNAP-25, fusion was not detected under anyconditions (Fig. 3a, right). In the absence of syt, very high SNAP-25concentrations (10–30 mM) were required to drive even modest fusion(B15% the level of fusion observed using preformed syntaxin–SNAP-25 heterodimers; Fig. 3a, right, filled squares), confirming

the observation of ref. 8. However, the addition of Ca2+–syt enhancedthe ability of SNAP-25 to rescue fusion even when low concentrationsof SNAP-25 were used (Fig. 3a, right, open circles). As a control, weused vesicles containing the copurified t-SNARE heterodimers toassess the maximum degree of stimulation by Ca2+–syt (Fig. 3a, left,open circles). At the highest concentration of SNAP-25 used insolution, we were able to recover B41% of the maximum valueobtained with vesicles containing the preformed t-SNARE heterodi-mers. These data suggest that syt aids in the assembly of functionalSNARE complexes.

We tested directly whether syt can assist in the assembly of SNAREproteins into complexes using the flotation assay described inFigure 1a. Vesicles containing syntaxin alone (syxr) were incubatedwith the soluble, cytoplasmic domain of syb (cd-syb) and solubleSNAP-25 in the presence or absence of syt plus either EGTA or Ca2+. Ifcd-syb or SNAP-25 bind reconstituted syntaxin, they will cofloat withthe syntaxin vesicles. In the absence of syt, little SNAP-25 or cd-sybbound syntaxin vesicles (Fig. 3b). However, coflotation of cd-syb andSNAP-25 was dramatically enhanced by Ca2+–syt.

The experiments described above were also performed using vesiclescontaining preformed syntaxin–SNAP-25 heterodimers to assess therole of syt in cd-syb assembly into SNARE complexes. Under theseconditions, in which SNAP-25 is maximally bound to syntaxin, thelevel of cd-syb recruitment is at its maximum with or without Ca2+–syt (Fig. 3b, last four lanes). These data indicate that although syt isrequired for the recruitment of SNAP-25 to the SNARE complex, therecruitment of cd-syb requires only the previously assembled t-SNAREcomplex. We note that in the case where we used vesicles thatharbored fully preformed t-SNARE heterodimers, greater levels of

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Figure 2 Ca2+–syt–t-SNARE interactions are crucial for Ca2+-stimulated membrane fusion. (a) Ca2+–syt stimulates neuronal SNARE–mediated membrane

fusion but not yeast SNARE–mediated membrane fusion. Syt was mixed with neuronal or yeast v- and t-SNARE vesicles in the presence of EGTA or Ca2+

and fusion was monitored at 37 1C. Inset, Coomassie-stained gel of the neuronal and yeast SNARE vesicles and syt used in the fusion and light-scattering

assays. (b) Syt aggregates both yeast and neuronal SNARE-bearing vesicles to the same extent in response to Ca2+. Syt was mixed with neuronal or yeast

v- and t-SNARE vesicles (containing 15% PS) and light-scattering (c.p.s.) was continuously recorded using a fluorometer. The same proportion of v- and

t-SNARE vesicles used in fusion assays was used for all light-scattering assays. Buffer (B), Ca2+ and excess EGTA (E) were added at the indicated times.

(c) Fusion of yeast SNARE vesicles is not stimulated by syt. The indicated [syt] was incubated with yeast v-SNARE vesicles and Sso1p vesicles plus the

indicated [Sec9c] in the presence of EGTA or Ca2+. Error bars show s.e.m. (d) The indicated [syt] was incubated with neuronal v- and t-SNARE vesicles in

the presence of either EGTA or Ca2+ and fusion was monitored as in a.

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cd-syb were not recruited despite the fact that these vesicles had agreater density of t-SNAREs. This finding suggests that some fractionof the preformed t-SNARE heterodimers are not active, as suggested ina recent study38.

Syt binds both t-SNAREs and PS. It was therefore possible thatCa2+–syt ‘pinned’ SNAP-25 to syntaxin vesicles by virtue of its PS-binding properties rather than by driving assembly of either SNAP-25or cd-syb onto syntaxin. In addition, it has been reported that cd-sybcan bind directly to phospholipids39, and hence it might cofloat withvesicles owing to interactions with membranes rather than t-SNAREs.We addressed these issues by omitting syntaxin from the vesicles andperforming the flotation assays as above using 15% PS/85% PCvesicles. Ca2+–syt cofloated with the PS-containing vesicles, butneither SNAP-25 nor cd-syb were detected in the sample (Fig. 3c).Thus, the recruitment of SNAP-25 and cd-syb shown in Figure 3b isnot due to Ca2+–syt pinning SNAP-25 to PS liposomes, but rather isdue to Ca2+–syt–mediated assembly of SNAP-25 and cd-syb intocomplexes with reconstituted syntaxin. We have been unable to detectinteractions between cd-syb and vesicles composed of PS and PC(A.B., M.C.C. and E.R.C., unpublished data).

The anionic lipid PS is absolutely required for Ca2+–syt stimulationof SNARE-mediated fusion32. Therefore, we tested whether PS is alsorequired for Ca2+–syt–mediated SNARE complex assembly. Omissionof PS strongly reduced Ca2+–syt–mediated assembly of SNAP-25 andcd-syb onto syntaxin-bearing vesicles (Fig. 3d). Thus, PS seems tofunction as a cofactor that allows Ca2+–syt to regulate t-SNAREstructure and function.

In the next series of experiments, we assayed the ability of Ca2+–sytto drive assembly of SNAP-25 onto reconstituted syntaxin, as a

function of time, in the absence of cd-syb. Ca2+ and syt stronglyfacilitated the assembly of t-SNARE heterodimers (Fig. 4). In parallelexperiments, Ca2+–syt had no effect on the binding of cd-syb toreconstituted syntaxin when SNAP-25 had been omitted (A.B., M.C.C.and E.R.C., unpublished data). These data suggest that, under theseconditions, Ca2+–syt first drives assembly of syntaxin–SNAP-25heterodimers, followed by recruitment of cd-syb.

We next carried out experiments to determine whether Ca2+–sytdoes in fact drive assembly of stable SNARE complexes. This wastested using two different approaches. First, assembly of SNAREproteins into SNARE complexes renders them resistant to cleavageby botulinum neurotoxins40. SNAP-25 that had been assembled into

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Figure 3 Ca2+–syt mediates assembly of SNARE complexes. (a) Ca2+–syt stimulates the ability of soluble SNAP-25 to drive fusion when added to

reconstituted syntaxin vesicles. Left, v-SNARE vesicles were mixed with t-SNARE vesicles harboring preformed syntaxin–SNAP-25 heterodimers in the

absence or presence of syt in either EGTA or Ca2+. Right, increasing concentrations of soluble SNAP-25 were added to syntaxin-bearing vesicles and

v-SNARE vesicles in the absence or presence of syt and Ca2+. Fusion data obtained in the presence of syt in EGTA is shown only for the highest

concentration of SNAP-25 tested. (b) Ca2+–syt enhances SNARE complex assembly. The indicated combinations of syntaxin-bearing vesicles (syxr; 15% PS,

85% PC), vesicles harboring t-SNARE heterodimers (Tr; 15% PS, 85% PC), cd-syb and soluble SNAP-25 were incubated in the presence or absence of syt

plus either EGTA (–) or Ca2+ (+). Samples were subjected to the flotation assay described in Figure 1a; proteins were separated by SDS-PAGE and stained

with Coomassie blue. Ca2+–syt increased the binding of SNAP-25 by a factor of 5.1 ± 1 and that of cd-syb by a factor 4.0 ± 0.9. (c) SNAP-25 and cd-syb

do not cofloat with protein-free vesicles. Syt, SNAP-25 and cd-syb were incubated with protein-free (Pf) vesicles composed of 15% PS and 85% PC and

subjected to flotation assays as in b. (d) PS is required for Ca2+–syt–stimulated assembly of SNARE complexes. Left, flotation assays were carried out

as in b with reconstituted syntaxin vesicles (syxr) containing either 100% PC or 15% PS and 85% PC in varying [Ca2+]. Right, bound SNAP-25 and cd-syb

were quantified by densitometry. AU, arbitrary units.

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Figure 4 Ca2+–syt enhances the rate of assembly of t-SNARE heterodimersin the absence of cd-syb. Syntaxin-bearing vesicles (syxr; 15% PS, 85% PC)

were incubated for the indicated times with SNAP-25 in the absence or

presence of syt in EGTA (–) or Ca2+ (+). Samples were then subjected to

the flotation assay described in Figure 1a and proteins were separated by

SDS-PAGE and stained with Coomassie blue.

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putative SNARE complexes by Ca2+–syt was largely protected fromcleavage by BoNT/E (Fig. 5). Second, the SNARE complex is extre-mely stable, with an off-rate of B4.2 � 10�18 s–1 (ref. 41). Hence, ifCa2+–syt drives assembly of SNARE complexes, they should remainintact even when syt has been removed using EGTA. Indeed, sytis readily dissociated from syntaxin vesicles upon chelation of Ca2+

with EGTA, but SNAP-25 and cd-syb recruited by the prior action of

Ca2+–syt remain in a complex with syntaxin (Fig. 5). Hence, Ca2+–sytdrives stable folding of SNARE proteins into bona fide toxin-resistantSNARE complexes.

In summary, the data described in this section provide the firstevidence for a direct action of Ca2+ and syt on the folding of SNAREproteins into complexes. Ca2+–syt can also act on preassembledt-SNARE complexes in a step after assembly of syntaxin–SNAP-25heterodimers31,32, and the structural basis for these effects remainsto be explored. However, the data described here demonstrate thatCa2+–syt influences the structure and function of isolated t-SNAREs.

Syt stimulates fusion of both membrane leafletsIn the final series of experiments, we tested whether Ca2+–sytstimulates hemifusion (mixing of phospholipids in the outer leafletonly) or full fusion (mixing of phospholipids in both the inner andouter leaflets) in the reconstituted fusion assay.

For these experiments we used dithionite, a reducing agent thatirreversibly quenches the fluorescence of NBD8,42,43. Owing to itshydrophilic properties, dithionite does not readily cross lipid bilayersand therefore selectively quenches NBD fluorescence in the outerleaflet of the vesicle, leaving NBD in the inner leaflet intact44. Aftertreating the outer leaflet with dithionite, the fluorescence increaseobserved in the fusion assay represents fusion of the inner leafletalone. Addition of the pore-forming peptide melittin45 allows dithio-nite to enter the lumen of the vesicles, where it can quench theremaining NBD fluorescence on the inner leaflet (Fig. 6a).

We found that at high concentrations, dithionite could enterv-SNARE vesicles to quench all NBD fluorescence, but the efficiencyof entry was dependent on how much syb had been reconstituted.When B100 syb molecules were reconstituted per vesicle (low-copy;Fig. 6b, open circles), a plateau in the dithionite titration wasapparent. This plateau (centered around 5 mM dithionite) isprobably due to quenching of NBD in the outer but not the

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–––

SNAP-25

BoNT/E

15% PS

Figure 5 SNARE complexes assembled by Ca2+–syt are resistant to cleavageby botulinum neurotoxin E. Syntaxin-bearing vesicles (syxr), soluble

SNAP-25 and cd-syb were incubated in the absence or presence of syt in

EGTA (–), Ca2+ (+) or Ca2+ followed by addition of excess EGTA to chelate

Ca2+ (±). Samples were subjected to the flotation assay described in

Figure 1a and material collected from the flotation step was treated with

BoNT/E where indicated. Samples were separated by SDS-PAGE and stained

with Coomassie blue. Once SNAP-25 became assembled into SNARE

complexes by the action of Ca2+–syt, it was largely protected from cleavage

by BoNT/E; the degree of protection was 70%–80%. As a control, soluble

SNAP-25, plus or minus syt, in EGTA or Ca2+, was treated with BoNT/E.

In all cases, cleavage of SNAP-25 by BoNT/E was complete.

LC syb vesicles

LC

HC syb vesicles

HC LC syb vesicles + detergent

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Figure 6 Syt stimulates fusion of both the inner and outer membrane leaflets ofSNARE-bearing vesicles. (a) Illustration depicting dithionite quenching of NBD

fluorescence in the presence and absence of melittin. Dithionite, a hydrophilic

reducing agent, quenches NBD fluorescence on the outer leaflet of the vesicle.

Subsequent addition of melittin, which forms pores, allows dithionite to quench

NBD on the inner leaflet. (b) The inner leaflet of low-copy syb vesicles is resistant to

dithionite quenching. High-copy (HC, see inset), low-copy (LC, see inset) or detergent-

solubilized low-copy syb vesicles were treated with the indicated [dithionite]. NBD

fluorescence (as a percentage of the maximum fluorescence) was plotted as a function

of [dithionite]. Error bars show s.e.m. Arrow indicates [dithionite] used in c and d.

Low- and high-copy syb vesicles were composed of 1.5% NBD-PS, 13.5% PS and

85% PC. (c) Dithionite (5 mM) does not quench the inner leaflet of NBD-containing vesicles. NBD fluorescence (Fl) of low-copy syb vesicles (1.5% NBD-PS,

13.5% PS and 85% PC) was monitored continuously at 37 1C. Dithionite, buffer and/or melittin were added as indicated in the key, at the times marked

by arrows. (d) Ca2+–syt stimulates SNARE-catalyzed fusion of outer and inner leaflets. Syt was mixed with syb vesicles (composed of 1.5% NBD-PS,

1.5% rhodamine-PE, 13.5% PS and 83.5% PC) and either vesicles harboring syntaxin–SNAP-25 heterodimers or protein-free (Pf) vesicles in the absence

(left) or presence (right) of dithionite. All reactions contained Ca2+.

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inner leaflet. Indeed, under these conditions,B60% of the NBD fluorescence wasquenched (Fig. 6b), which corresponds tothe ratio of outer to inner leaflet surfacearea of a vesicle. Therefore, low-copyv-SNARE vesicles and 5 mM dithionite wereused in all further experiments. We note thatwhen high-copy v-SNARE vesicles were used(B1,000 syb per vesicle; Fig. 6b, filledsquares), the addition of 5 mM dithioniteresulted in a 475% drop in fluorescence,suggesting that these vesicles are more leakythan the low-copy v-SNARE vesicles.

In another series of control experiments, we added dithionite to low-copy v-SNARE vesicles and observed roughly the same drop in NBDfluorescence as in Figure 6b (Fig. 6c, filled upright triangles). Sub-sequent addition of melittin, which forms pores in the vesicles, makingthem permeable to dithionite, resulted in complete quenching of NBDon the inner leaflet without further addition of dithionite (Fig. 6c,open squares). Thus, dithionite was not limiting in this experiment.These data demonstrate that the inner leaflet is resistant to 5 mMdithionite, and this concentration was used in all subsequent assays.

We then determined whether Ca2+–syt stimulates hemifusion or fullfusion. Fluorescence increases, observed in the fusion assay afterquenching of the signal from the outer leaflet, represent fusion ofthe inner leaflet and hence report complete fusion of the v- andt-SNARE vesicles. First, in a positive control experiment, syt stimulatedSNARE-catalyzed membrane fusion in response to Ca2+ (Fig. 6d).Parallel samples were treated with 5 mM dithionite to destroy the NBDsignal on the outer leaflet and the fusion assay was repeated. There wasa B50%–60% fluorescence drop in all samples owing to selectiveloss of the fluorescence signal from outer leaflets of the v-SNAREvesicles (Fig. 6d, right). In the example shown, Ca2+–syt caused similarincreases in inner leaflet mixing as compared to inner plus outerleaflet mixing (1.4- to 1.6-fold stimulation). Furthermore, the rate ofinner as compared to inner plus outer leaflet mixing was the same(Supplementary Fig. 2 online), suggesting that hemifusion eitherdid not occur or was not rate limiting under these experimentalconditions. Thus, SNAREs drive complete fusion—both inner andouter leaflets mix—and this reaction is enhanced by Ca2+–syt. Wenote that dithionite has a short half-life in water. In Figure 6d (right),we allowed the dithionite to become inactivated before the fusionassay was carried out. Consequently, addition of detergent at the endof the measurement did not result in further quenching of theNBD signal.

DISCUSSIONA hallmark of synaptic vesicle exocytosis is the speed of this processand its strict regulation by Ca2+ (ref. 46). Although SNARE proteinsare required for exocytosis3 and are thought to be the minimal fusionmachinery sufficient for membrane fusion in vitro8, regulation byCa2+ seems to be provided by the Ca2+-sensing protein syt31.A handful of syt-effector interactions (for example, with PS andt-SNAREs) have been postulated to be crucial for membranefusion23,28,30,33,47. In particular, the interaction of the Ca2+ sensorfor exocytosis with the core of the fusion machinery is conceptuallyappealing as a coupling step in secretion; however, the relevance ofsyt–t-SNARE interactions in membrane fusion has been questionedand direct experiments to address this issue have been lacking48,49.In this study, we have tested directly the functional significance ofsyt–t-SNARE interactions during fusion.

We approached this question by studying fusion mediated byneuronal and yeast SNARE complexes. Ca2+–syt stimulated mem-brane fusion only between vesicles that contained the t-SNAREs that itbinds to (that is, neuronal but not yeast t-SNAREs). All other Ca2+–syteffector interactions in this defined fusion reaction remained intact(for example, binding to PS26,34, oligomerization35 and aggregation ofvesicles). Moreover, we have shown that Ca2+–syt, in addition tobinding and acting on preformed SNARE complexes, acts on indivi-dual t-SNAREs to facilitate their assembly into heterodimers inresponse to Ca2+. Once Ca2+–syt assembles SNARE proteins in vitro,this complex is stable and does not require the continuous presence ofsyt (Fig. 5).

Previously, it has been shown that PS is an essential effectorfor Ca2+–syt stimulation of fusion32. Therefore, we asked whetherPS has a role in Ca2+–syt–mediated SNARE assembly or whetherPS is required by syt only after the SNARE complex is assembled.In the presence of Ca2+–syt–PS, SNAP-25 and cd-syb were effi-ciently recruited onto syntaxin vesicles; omission of PS largelyabrogated this. These data suggest that PS is a cofactor forCa2+–syt–mediated SNARE assembly as well as for stimulatingSNARE-catalyzed membrane fusion after assembly of t-SNAREheterodimers32.

Recent studies suggest that SNARE-catalyzed membrane fusiontransits through a hemifusion intermediate43,50–52, ultimately culmi-nating in full fusion. However, the question of whether syt stimulateshemifusion or full fusion had not been addressed. In the final set ofexperiments, we demonstrated that Ca2+–syt promotes SNARE-mediated fusion of both the inner and outer leaflets and hencepromotes full fusion. Under our experimental conditions, we didnot see evidence for a hemifusion intermediate in either the absence orpresence of Ca2+–syt, but we note that others have reported conditionsunder which hemifusion is apparent43,50–52.

On the basis of the data presented in this study, we propose a modelin which Ca2+–syt–PS complexes bind and alter the structure ofsyntaxin and SNAP-25 so that they fold into heterodimers (Fig. 7).The t-SNARE heterodimer then forms a high affinity receptor for thev-SNARE syb, in agreement with previous studies showing that thev-SNARE syb binds syntaxin with higher affinity in the presence ofSNAP-25 (ref. 53). Ca2+–syt–PS then drives structural rearrangementsin partially or fully assembled SNARE complexes to trigger theopening and dilation of fusion pores23,54–56. Dilation could be a resultof Ca2+–syt–mediated lateral separation of SNARE proteins thatinitially line the fusion pore57.

In conclusion, the data reported here provide direct evidence for theidea that Ca2+–syt acts directly on t-SNAREs to alter their structureand function. Now, a key issue is to understand the effect of Ca2+–syton syntaxin and SNAP-25 after Ca2+–syt has driven their assemblyinto heterodimers.

C2BC2A

Ca2+ –syt–PS

Regulates assembly of

SNARE complexes

Drives structuraltransitions in SNAREs

syt

syb

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1Ca2+ –syt Ca2+ –syt

32

Expansionof pore

Opening offusion pore

Figure 7 Model depicting the role of Ca2+–syt in SNARE-catalyzed membrane fusion. A complex of syt,

Ca2+ and PS regulates assembly of SNARE complexes (1); Ca2+–syt then drives structural transitions

in partially or fully assembled SNARE complexes to trigger fusion pore opening (2), and Ca2+–syt

regulates fusion-pore dilation (3)23,54. Model is modified and reproduced with permission from ref. 17.

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METHODSPlasmids and protein purification. Complementary DNA encoding rat syt I19

was provided by T.C. Sudhof (University of Texas Southwestern Medical

Institute) and the Asp374 mutation was corrected by substitution with

a glycine residue58. The cytoplasmic domain of syt was used throughout

this study and corresponds to residues 96–421. This fragment of syt was

subcloned into a pTrc-His vector (Invitrogen), expressed in Escherichia coli and

purified as described in refs. 32,59, with modifications (for details see

Supplementary Methods).

Plasmids to generate recombinant full-length syb-2 (pTW2), the cytoplasmic

domain of syb (cd-syb; residues 1–94; pET-rsybCD) and the full-length t-SNARE

heterodimer (syntaxin-1A and SNAP-25; pTW34), were provided by J.E. Roth-

man (Columbia University) and proteins were expressed and purified as

described in refs. 8,31. We also generated individual t-SNAREs. To this end,

cDNAs encoding full-length SNAP-25 (ref. 60) (provided by M.C. Wilson,

University of New Mexico) and full-length syntaxin-1A21 (provided by R.H.

Scheller, Genentech) were subcloned into a pTrc-His vector (Invitrogen),

resulting in N-terminal His6 tags, and purified as above (see also Supplementary

Methods). We note that syntaxin and SNAP-25 that were purified as individual

proteins have a higher molecular weight than their counterparts in the co-

expressed heterodimers (Fig. 3b). This is because use of the pTrcHis vector

results in 42-residue tags on the N termini of syntaxin and SNAP-25. In the

heterodimers, syntaxin is untagged and SNAP-25 has a 12-residue tag.

Recombinant Sso1p (pJM285; residues 1–290; yeast syntaxin homolog),

Sec9c (pJM418; residues 401–651; yeast SNAP-25 homolog) and Snc2p

(pJM81; residues 1–115; yeast syb homolog) were provided by J. McNew (Rice

University); proteins were expressed and purified as described for neuronal

SNAREs, with modifications (see Supplementary Methods).

Preparation of protein-free and SNARE-bearing vesicles. All lipids were

obtained from Avanti Polar Lipids. Reconstitution of v-SNARE and t-SNARE

vesicles was carried out as previously described8,31. Briefly, v-SNAREs were

reconstituted using a lipid mix composed of 82% 1-palmitoyl, 2-oleoyl

phosphatidylcholine (PC), 15% 1,2-dioleoyl phosphatidylserine (PS), 1.5%

N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidylethanola-

mine (NBD-PE, donor) and 1.5% N-(lissamine rhodamine B sulfonyl)-1,2-

dipalmitoyl phosphatidylethanolamine (rhodamine-PE, acceptor). t-SNAREs

were reconstituted in 85% PC and 15% PS (mol/mol). When PS was omitted,

PC was increased to 97% and 100% for v- and t -SNARE vesicles, respectively.

v-SNARE (syb or Snc2p) and t-SNARE (syntaxin–SNAP-25, syntaxin alone or

Sso1p alone) vesicles were reconstituted to give B100 copies and B80 copies

per vesicle, respectively, as described in ref. 31. At B80 copies per vesicle, the

t-SNARE concentration in the fusion assay is B3 mM. High-copy (B1,000 syb

per vesicle) and low-copy (B100 syb per vesicle) vesicles were prepared as

described in refs. 8,31. Yeast t-SNARE heterodimer vesicles (Sso1p–Sec9c) were

made by incubating Sec9c (7 mM) with vesicles bearing reconstituted Sso1p for

1 h at room temperature with shaking. Protein-free vesicles were prepared as

described previously31.

Flotation assays. Flotation assays were carried out as described previously, with

modifications31,32. Briefly, v- or t-SNARE vesicles were mixed with syt (10 mM),

SNAP-25 (10 mM), cd-syb (20 mM) or a combination of these in either 0.2 mM

EGTA or 1 mM Ca2+ and floated through an Accudenz gradient. Floated

material was collected, subjected to SDS-PAGE and stained with Coomassie

blue (or western blotted; Fig. 1c) to monitor binding (Supplementary

Methods). All Coomassie stained gels and western blots presented throughout

this study are representative examples from at least three trials.

Cleavage of SNAP-25 by botulinum neurotoxin E. Flotation assays were

carried out as described above under the conditions indicated in Figure 5. After

flotation, each sample was split into two; one half served as a control and the

other half was treated with BoNT/E (65 nM) at 37 1C for 15 min. Soluble

SNAP-25 (1.5 mM) served as a control to monitor BoNT/E activity under the

indicated conditions. Samples were subjected to SDS-PAGE and stained with

Coomassie blue (Supplementary Methods).

Fusion assays. Fusion assays were carried out as described previously31 (for

details see Supplementary Methods). Briefly, v- and t-SNARE vesicles were

mixed in the absence or presence of syt, in either 0.2 mM EGTA or 1 mM Ca2+.

NBD fluorescence was monitored for 2 h at 37 1C. In all figures, data from

fusion assays are representative examples from at least three independent trials.

Light-scattering. The indicated vesicles plus syt (10 mM) were continuously

mixed in a quartz cuvette (total volume of 600 ml) in a fluorometer (QM1;

Photon Technology International) and 901 light-scattering (400 nm light) was

monitored as a function of time. The intensity of scattered light was read out as

counts per second (c.p.s.). Buffer, Ca2+ (1 mM final concentration) and EGTA

(2 mM final concentration) were added sequentially as indicated by arrows.

The initial light-scattering signal was slightly lower for the yeast SNARE

liposomes and was offset by o30%. Figure 2b shows a representative example

from at least three independent trials.

Dithionite quenching assays. NBD fluorescence (460 nm excitation, 535 nm

emission) from 5 ml of the indicated v-SNARE vesicles (1.5% NBD-PS, 13.5%

PS and 85% PC; total volume of 75 ml) was measured in a plate reader

(Molecular Probes SpectraMax Gemini) at 37 1C until the NBD fluorescence

stabilized. Dithionite was then added and the reaction was monitored until the

signal stabilized. The fluorescence intensity of dithionite-treated samples was

normalized using spectra from samples lacking dithionite. When used, melittin

was added to a final concentration of 1:20 melittin/lipid. Figure 6 shows

representative quenching experiments from three independent trials. Fusion

assays to monitor inner-leaflet dequenching were carried out as described

above, but using v-SNARE vesicles (composed of 1.5% NBD-PS, 1.5%

rhodamine-PE, 13.5% PS and 83.5% PC) that were preincubated with 5 mM

dithionite at room temperature for 30 min.

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSWe thank members of E.R.C.’s laboratory for discussions and comments. Thisstudy was supported by grants from the US National Institutes of Health(NIGMS GM 56827 and NIMH MH61876) and from the American HeartAssociation (0440168N) to E.R.C. A.B. and M.C.C. are supported by an AmericanHeart Association predoctoral fellowship. W.C.T. was supported by a postdoctoralNational Research Service Award from the US National Institutes of Health.E.R.C. is an Investigator of the Howard Hughes Medical Institute.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/nsmb/

Reprints and permissions information is available online at http://npg.nature.com/

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