SNARE-MEDIATED MEMBRANE FUSION - UNAMpt7mdv.ceingebi.unam.mx/computo/pdfs/cursosviejos/bcelular/SNARE… · SNARE-cleaving neurotoxins do not affect vesicle docking at the synapse

98 | FEBRUARY 2001 | VOLUME 2 www.nature.com/reviews/molcellbio

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In eukaryotic cells, molecules need to be delivered totheir correct intracellular destinations without compro-mising the structural integrity of cellular compart-ments. To achieve this, transport vesicles bud from anintracellular donor organelle and then target, dock andfuse with an acceptor organelle. Membrane fusion isalso involved in organelle inheritance during mitosisand in cell growth or division, which require membraneaddition. In the nervous system, membrane fusion is anessential step in chemical synaptic transmission becauseneurotransmitter-filled PRESYNAPTIC vesicles fuse in a cal-cium-dependent manner with the plasma membraneto release their content into the SYNAPTIC CLEFT.Furthermore, the fusion of POSTSYNAPTIC-membrane-receptor-containing vesicles with the plasma membraneimplicates the membrane fusion machinery in long-term modulation of synaptic strength, which is impor-tant for memory and learning1.

A vesicle fusion event involves many coordinatedsteps. Before fusion, a vesicle is transported to its spe-cific target membrane and docked or tethered there2. Itthen goes through several ‘priming’ events to prepare itfor release3. A fusion trigger — Ca2+ in many traffickingevents4–7 — then directs fusion to proceed to comple-tion. To achieve this, the cellular fusion machinerymust overcome the repulsive ionic forces and dissipatethe hydration between the two lipid bilayers. Thisreview focuses on the SNARE family of proteins, asthey have been implicated as the conserved core pro-tein machinery for all intracellular membrane fusionevents. It summarizes the significant progress made

during the past three years in understanding the mech-anism of SNARE function.

The SNARE superfamilySNARE (soluble NSF attachment protein receptor whereNSF stands for N-ethyl-maleimide-sensitive fusion pro-tein) proteins have been implicated as central in most, ifnot all, intracellular membrane trafficking events studiedso far. The synaptic proteins syntaxin (STX1)8, SNAP-25(25 kDa synaptosome-associated protein)9 and VAMP10

(vesicle-associated membrane protein, also called synap-tobrevin11; for example see VAMP1) were the firstSNAREs to be discovered.Yeast proteins that are essen-tial for secretory function, including some SNAREs,were independently discovered in genetic screens12. Bothsyntaxin and VAMP are anchored to the membrane by acarboxy-terminal transmembrane domain, whereasSNAP-25 is peripherally attached to the membrane byPALMITOYLATION of four cysteine residues in the centralregion of the protein. SNAREs were originally dividedinto v-SNAREs and t-SNAREs according to their vesicleor target membrane localization13. However, to avoidambiguity in the case of homotypic membrane fusion,SNAREs have been reclassified as R-SNAREs (arginine-containing SNAREs) or Q-SNAREs (glutamine-con-taining SNAREs), based on the identity of a highly con-served residue14. The hallmark of all SNARE proteins isthat they contain conserved heptad repeat sequences intheir membrane-proximal regions that form coiled-coilstructures.

More than a hundred other SNARE proteins from

SNARE-MEDIATED MEMBRANE FUSIONYu A. Chen* and Richard H. Scheller‡

SNARE proteins have been proposed to mediate all intracellular membrane fusion events.There are over 30 SNARE family members in mammalian cells and each is found in a distinctsubcellular compartment. It is likely that SNAREs encode aspects of membrane transportspecificity but the mechanism by which this specificity is achieved remains controversial.Functional studies have provided exciting insights into how SNARE proteins interact with eachother to generate the driving force needed to fuse lipid bilayers.

*Renovis Inc., 747 FiftySecond Street, Oakland,California 94609, USA.‡Department of Molecularand Cellular Physiology,Howard Hughes MedicalInstitute, Stanford University,Stanford, California 94305,USA.e-mails:[email protected];[email protected] to R.H.S.

PRESYNAPTIC

Pertaining to the neuron thattransmits impulses to a synapse.

SYNAPTIC CLEFT

The extracellular space,typically ~20 nm across, thatseparates the outer membraneof the presynaptic nerve endingfrom the postsynapticmembrane of the receiving cellin a synapse.

POSTSYNAPTIC

Pertaining to the neuron or themuscle cell that is on theefferent side of a synapse, whichtransduces signals away fromthe synapse.

© 2001 Macmillan Magazines Ltd

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | FEBRUARY 2001 | 99

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Fig.1 online), which might indicate that they haveselective functional involvement in specific intracellu-lar trafficking steps.

Specificity of membrane traffickingIt was initially believed that specific interactions of v-SNAREs and t-SNAREs confer specificity on intracellu-lar membrane trafficking (BOX 1). Intuitively, however,the specificity of membrane trafficking is most probablydefined at the vesicle targeting and tethering stages, dur-ing which vesicles are captured and tethered by longextended proteins from the target membrane2. SmallGTPases of the Rab family have been proposed to beimportant in the early stage of vesicle targeting and teth-ering, and many of them have been found to localize todistinct cellular compartments (see the review by Zerialand McBride on page 107 in this issue). Therefore, it islikely that Rab proteins are important in determiningvesicular transport specificity.

Most of the recent studies indicate that SNAREs mightmediate membrane fusion and not docking. Is it possible,then, that the SNARE-mediated fusion specificity issuperimposed on the Rab-mediated docking specificityto make the system even more reliable? A large number ofSNARE homologues localize to specific membrane com-partments throughout the secretory pathway, whichwould be wasteful if they all had the same function of fus-ing two attached membranes. Although some SNAREscan function in several trafficking steps and substitute forother SNAREs17, it is not clear whether this is generallytrue. In the CRACKED PC12 CELL SYSTEM, in which fusionbetween DENSE CORE GRANULES and the plasma membranecan be measured, soluble cognate SNAREs rescued orcompeted more successfully than non-cognate SNAREs,showing a high degree of SNARE specificity16. In recentliposome fusion experiments, both topological specificity(two t-SNAREs on one membrane and one v-SNARE onthe other membrane)18 and pairing specificity19 wereobserved. However, a certain degree of promiscuity wasalso observed, as any R-SNARE could fuse with plasma-membrane Q-SNAREs19. So, it seems that the intrinsicphysio-chemical properties of the SNARE proteins, aswell as upstream targeting and tethering factors, encodeaspects of specificity in intracellular membrane transport.

diverse organisms have been discovered15. On thebasis of sequence homology and domain structure,the known mammalian SNAREs have been catego-rized as members of the syntaxin, VAMP or SNAP-25families. Most of them are found in specific cellularcompartments15,16 (FIG. 1 and hyperlinked version of

PALMITOYLATION

Covalent attachment of apalmitate (16-carbon saturatedfatty acid) to a cysteine residuethrough a thioester bond.

PC12 CELLS

A clonal line of rat adrenalpheochromocytoma cells whichrespond to nerve growth factorand can synthesize, store andsecrete catecholamines, muchlike sympathetic neurons. PC12cells contain small, clearsynaptic-like vesicles and largerdense core granules.

CRACKED PC12 CELL SYSTEM

Exocytosis assay in which PC12cells are mechanicallypermeabilized by a ballhomogenizer, and secretion of[3H] noradrenaline from densecore granules is reconstitutedand measured.

SNAP-25SNAP-23

STX5 STX11

CCP

CCV

Plasmamembrane

Earlyendosome

Nucleus

Lysosome

RER

SER

Golgi

TGN

IC

Lateendosome

VAMP1VAMP2

STX7STX8VAMP3VAMP8

STX13

VAMP1VAMP2

DCV

STX6 STX10 STX11 STX16

STX17

Membrin

STX18STX5SEC22BBET1

VAMP7VAMP8

STX7STX8

STX7STX8

BET1

SEC22BYKT6

STX1STX2STX3STX4 STX3

VAMP2SNAP-23 V

VAMP5

Membrin

STX5VAMP4SNAP-29

GOS28

VTI1

Figure 1 | Subcellular localization of mammalian SNAREs. The mammalian SNAREs thathave been studied so far localize to distinct subcellular compartments in the secretorypathway. (Red, syntaxin family; blue, VAMP family; green, SNAP-25 family; black, others. CCP,clathrin-coated pit; CCV, clathrin-coated vesicles; DCV, dense core vesicles; IC, intermediatecompartment; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum;SNAP-25, 25 kDa synaptosome-associated protein; TGN, trans-Golgi network; V, vesicles;VAMP, vesicle-associated membrane protein.)

Box 1 | The SNARE hypothesis

The SNARE (soluble NSF attachment protein receptor, where NSF stands for N-ethyl-maleimide-sensitive fusionprotein) hypothesis was proposed in 1993, before most of the current knowledge became available, as the firstworking model to explain vesicle docking and fusion in molecular terms13. It postulated that each type of transportvesicle has a distinct v-SNARE that pairs up with a unique cognate t-SNARE at the appropriate target membrane,and that this specific interaction docks the vesicles at the correct membrane, with the subsequent dissociation of theSNARE complex by the ATPase activity of NSF driving membrane fusion.

Although the biochemical activity of α-SNAP and NSF — dissociating the SNARE complex — has not beendisputed, the specific roles of α-SNAP and NSF in this process have since been revised94. The current view is that NSFacts as a chaperone to reactivate SNAREs after one round of fusion, instead of directly driving fusion (FIG. 4).

The role of SNAREs in docking, proposed by the SNARE hypothesis, was also challenged by the finding thatSNARE-cleaving neurotoxins do not affect vesicle docking at the synapse95, and that SNARE-deficient flies have anincreased, not decreased, number of morphologically docked vesicles96,97. Current evidence indicates that smallGTPases of the Rab protein family might be crucial for docking or tethering vesicles (see the review by Zerial andMcBride on page 107 in this issue), whereas SNARE pairing is involved at a later step of membrane fusion. Moreover,it is the assembly, not disassembly, of the core complex that probably drives lipid fusion (FIG. 4).



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ports the hypothesis that formation of the SNARE com-plex fuses two membranes by bringing them into closeapposition rather than simply docking them (FIG. 2a).

As might be expected for a complex that is so stable,ATP is needed to dissociate it into monomeric compo-nents. Disassembly is carried out by two proteins, theATPase NSF and an adaptor protein, α-SNAP, (solubleNSF attachment protein), which were initially discov-ered as essential factors in a GOLGI TRANSPORT ASSAY28,29.Several structural studies of α-SNAP, NSF and theSNARE–SNAP–NSF complex25,30–35 indicate a possiblemodel in which the ATP-dependent enzymatic activityof NSF might impart a rotational shear to dissociate thecore complex.

SNARE domains outside the helical bundleSNARE domains that are not part of the core complexinclude the amino-terminal domain of syntaxin, the cen-tral palmitoylated region of SNAP-25, the amino-termi-nal proline-rich region of VAMP, and the transmem-brane domains of syntaxin and VAMP (FIG. 3). The longamino-terminal domain of syntaxin forms a three-helixbundle36,37 that competes with the VAMP and SNAP-25coils for binding of its own carboxy-terminal coil (form-ing the ‘closed’ conformation of syntaxin)38,39. So, asexpected, it decreases the rate of ternary SNARE complexformation in solution40 and the rate of fusion of syntheticliposomes that carry SNARE proteins41. The amino-ter-minal domain of syntaxin, along with the coil domain, isalso required for the interaction between syntaxin and n-Sec1 (REFS. 24, 39), which is also called Munc-18 (REF. 42).The chaperone protein n-Sec1 binds to the closed con-formation of syntaxin and, after a conformationalchange, probably opens it up to facilitate SNARE com-plex formation43,44 (FIGS 3, 4). So, the amino terminus ofsyntaxin is probably important for regulation in vivo.

The SNAP-25 loop region between the two coildomains is dispensable for SNAP-25 fusogenic activi-ty16,41,45. However, it is probably crucial for ensuringrapid synaptic transmission by generating a highlocal concentration of the two required SNAP-25coils by covalently linking them and attaching themto the plasma membrane. In the case of non-synapticfusion events, in which speed might be less impor-tant, the two SNAP-25-like coils are sometimes con-tributed by separate proteins46.

The function of the proline-rich amino-terminal~24 amino acids of VAMP (FIG. 3) is not clear. Thisdomain is only about 50% homologous betweenVAMP1 and VAMP2, but the proline-rich character ismaintained47. Although this domain is crucial for inhi-bition of EXOCYTOSIS by synthetic VAMP peptides inAplysia californica48, a VAMP coil without the proline-rich domain inhibits exocytosis efficiently in crackedPC12 cells16. Moreover, Fab FRAGMENTS of antibodiesdirected against the proline-rich region of VAMP2 didnot significantly affect exocytosis in adrenal CHROMAFFIN

CELLS49, indicating that this domain of VAMP is probablynot directly involved in membrane fusion. But it is notyet known whether it could be important for interact-ing with regulators of VAMP function in vivo.

SNARE core complexBiochemical studies have shown that the solublecoiled-coil-forming domains of recombinant syntaxin,SNAP-25 and VAMP form an extremely stablecomplex20,21. This ‘core complex’ is resistant to SDSdenaturation22, protease digestion20–22 and CLOSTRIDIAL

NEUROTOXIN cleavage22, and is heat stable up to ~90 oC(REF. 23). The affinity within the ternary complex ismarkedly higher than the pairwise binary affinitiesbetween the three proteins22,24. However, the exact dis-sociation constant of the ternary interaction has notyet been determined.

The crystal structure of the neuronal SNARE corecomplex (BOX 2) shows that one coil of syntaxin andVAMP, and two coils of SNAP-25 intertwine to form afour-stranded coiled-coil structure (FIGS 2, 3). This con-firms several elegant structural studies that predict a par-allel arrangement (with all amino termini at one end ofthe bundle) of the core complex25–27, which strongly sup-

b

...

......

Q

Q R

VI

IV

.

..

.

.

R

R D

R

D

K

Q

Q

Q

a b

VAMP

SyntaxinSNAP-25

Figure 2 | SNARE proteins form a four-helical bundle complex that drives membranefusion. a | VAMP (blue) on the vesicle interacts with syntaxin (red) and SNAP-25 (green) on theplasma membrane to form a four-helix bundle that zips up concomitant with bilayer fusion. b | The backbone of the SNARE complex is shown on the left52, with the central ionic layer (red)and 15 hydrophobic layers (black) that mediate the core interactions highlighted. Top-downviews of side-chain interactions are shown on the right, with the four SNARE helices shown asribbons. The ball-and-stick structures represent the indicated amino acids; the dotted linesrepresent hydrogen bonds or salt bridges that stabilize interactions between SNAREs. Q-SNAREs and R-SNAREs are characterized by a glutamine (Q) or arginine (R) residue,respectively, in the central layer of the SNARE complex. (SNARE; soluble NSF attachmentprotein receptor, where NSF stands for N-ethyl-maleimide-sensitive fusion protein; SNAP-25,25 kDa synaptosome-associated protein; VAMP, vesicle-associated membrane protein.)

DENSE CORE GRANULES

Large diameter (80–200 nm)secretory vesicles that have highelectron density underelectronmicroscopy. Theyusually contain neuropeptidesor catecholamines.

CLOSTRIDIAL NEUROTOXINS

Bacterial toxins that potentlyblock neurotransmitter releasethrough theirmetalloproteolytic activitydirected specifically towardsSNARE proteins. Includesbotulinum neurotoxins andtetanus toxin.

GOLGI TRANSPORT ASSAY

In vitro reconstitution assayconsisting of isolated Golgistacks, Mg-ATP and cytosol,where transport-coupledglycosylation is monitored.



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served between species than between different SNAREfamily members, it is likely that they are important forspecific regulation of the particular fusion reactionthat they are involved in.

Do SNAREs mediate membrane fusion?The ‘zipper’ model of SNARE function postulates thatthe SNARE core complex ‘zips’ from the membrane-dis-tal amino termini to the membrane-proximal carboxyltermini, and the formation of the stable SNARE com-plex overcomes the energy barrier to drive fusion of thelipid bilayers25,26. This hypothesis has gained substantialexperimental support during the past few years. InDrosophila melanogaster, a temperature-sensitive muta-tion in syntaxin that abolishes formation of the SNAREcomplex rapidly blocks exocytosis of docked vesicles55.In the cracked PC12 cell system56, SNAP-25 could bemanipulated by inactivating endogenous SNAP-25 withBOTULINUM NEUROTOXIN E, and then adding back a soluble

The transmembrane domains of syntaxin andVAMP have been shown to contribute to SNARE bind-ing in vitro21,50,51. It was hypothesized that the trans-membrane domains might form α-helices that contin-ue into the cytoplasmic coiled-coil bundle52. However,the introduction of a helix-breaking proline residuebetween the cytoplasmic and transmembrane domainsof VAMP did not significantly affect its ability to fuseliposomes53. Furthermore, both syntaxin and VAMPtransmembrane domains can be substituted with bilay-er-spanning POLYISOPRENOID synthetic anchors withoutaffecting liposome fusion54. So, at least in the artificialliposome system, the interaction between the trans-membrane domains of syntaxin and VAMP does notseem to be crucial for fusion.

These precise domain organizations of the threeneuronal SNAREs are not always preserved in othermembers of the SNARE family. However, as thesenon-coiled-coil domains of SNAREs are more con-

EXOCYTOSIS

The discharge by a cell ofintracellular materials into theextracellular space throughfusion of vesicles (containingthese materials) with theplasma membrane.

Fab FRAGMENT

Antigen-binding fragment of animmunoglobulin molecule. It isused when multimerization ofantibodies caused by their Fcdomains is not desirable.

CHROMAFFIN CELLS

They arise from the sameprecursors as sympatheticneurons, and can synthesize,store and secretecatacholamines. They are foundin all vertebrates, at variousbodily locations but especiallyin the medulla of the adrenalgland.

POLYISOPRENOID

Synthetic molecule consistingof varying numbers ofbranched five-carbon-atommoieties.

BOTULINUM NEUROTOXIN E

Clostridial neurotoxin thatcleaves SNAP-25 carboxy-terminal coil.

Core complex

n-Sec1–syntaxin complex

Coil Coil CoilPP TM TMHa Hb Hc CoilCCCC

N N N

SNAP-25 VAMP Syntaxin

n-Sec1

Syntaxin

Figure 3 | SNARE domain structures and the interaction between syntaxin and its chaperone protein n-Sec1. Theamino-terminal domain of syntaxin forms a three-helix bundle (red) that binds to its carboxy-terminal coil domain (purple),forming the closed conformation (right), which is bound and stabilized by n-sec1 (middle)39. A conformational change thenoccurs to allow dissociation of n-sec1 and the opening up of syntaxin, facilitating core complex formation. The coil domains ofsyntaxin, SNAP-25 and VAMP form the four-helix bundle core complex (left)52. In addition to the coil domain, VAMP harbours aproline-rich amino-terminal domain (PP) and SNAP-25 harbours a central domain that contains four palmitoylated cysteineresidues (CCCC). (SNAP-25, 25 kDa synaptosome-associated protein; TM, transmembrane domain; VAMP, vesicle-associatedmembrane protein.)

Box 2 | Crystal structure of the core complex

Crystallization of the SNARE core complex revealed a four-helix bundle structure52 (FIGS 2, 3). Like other coiled-coilstructures, the residues residing at ‘a’ or ‘d’ positions on a heptad helical wheel contribute to the hydrophobic coreinteractions that are important in stabilizing the structure. These residues are the most conserved residues in theSNARE family14. Interestingly, the Gln and Arg residues mentioned above (Arg from VAMP and three glutamines fromsyntaxin and the SNAP-25 amino- and carboxy-terminal coils) form a central ionic interaction layer (the zero layer) inthe otherwise hydrophobic core of the SNARE complex52 (FIG. 2b). Intrigued by the extreme conservation of these Glnand Arg residues through evolution, many researchers have looked into the exact role of these residues. In yeast, Gln toArg substitutions were found to result in drastically reduced secretion98,99, whereas an Arg to Gln mutation did notcause any abnormality by itself but rendered the core complex more sensitive to additional mutations98. In PC12 cells,mutating the Gln residue in the SNAP-25 carboxy-terminal coil to alanine (Q174A) only caused a slight decrease inexocytosis45. In adrenal chromaffin cells, overexpression of Q174L SNAP-25 selectively affected the sustained phase ofexocytosis and not the exocytic burst100, indicating that this ionic layer might be involved in facilitating SNARE complexdisassembly or their initial contacts. It is likely that the 3Q:1R ratio contributes to the correct SNARE bindingspecificity, as a Q→R mutation can be rescued by a mirror R→Q mutation in the opposite helix in the SNAREcomplex98,99. This unusual layer might also enforce the correct register during SNARE pairing.



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complexes did not affect content mixing of thevacuoles60. The conclusion that SNAREs do not mediatemembrane fusion was also drawn from studies usingthe SEA URCHIN EGG FUSION SYSTEM, in which Ca2+ ions werefound to dissociate SNARE complexes with no detri-ment to the fusion process61,62.

If SNAREs are not involved in membrane fusion,which proteins actually mediate the bilayer fusion reac-tion? Two of the downstream factors that have beenidentified in the yeast vacuole system are calmodulin5

and protein phosphatase 1 (PP1)63. In mammalian sys-tems, calmodulin has been reported to regulate the exo-cytic machinery64–67 but there has been little indicationthat mammalian PP1 is crucial for membrane trans-port. In fact, it seems unlikely that dephosphorylationcould be the final Ca2+-triggered event in synaptic vesi-cle fusion, given that neuronal exocytosis occurs withina millisecond68. The most likely event that could occurwithin such a short time, other than lipid rearrange-ment, is probably a protein conformational change. Thezipping of the SNARE core complex would be morecompatible with the timescale of fusion that is requiredby neurons. Although it is possible that the molecularmachinery mediating bilayer fusion differs fundamen-tally between organisms (such as yeast and mammals)and/or between different transport steps (such as presy-naptic exocytosis and homotypic vacuole fusion), it ismore likely that the principle is conserved. We favourthe hypothesis that SNARE proteins do mediate yeastvacuole fusion, and that dephosphorylation by PP1 issimultaneously required to facilitate SNARE-catalysedlipid fusion in this system.

SNAP-25 coil to rescue toxin-inhibited exocytosis aftertoxin washout45. In such a rescue assay, mutating thehydrophobic residues along the carboxy-terminal coilof SNAP-25 most severely affected the rescue, confirm-ing that the formation of the core complex is crucial forSNARE function16,45. Furthermore, the irreversibleassembly of the SNARE core complex occurred onlyafter the arrival of Ca2+ and could not be experimentallyuncoupled from the membrane fusion process45.Consistent with this result, electrophysiological kineticanalysis of exocytosis in adrenal chromaffin cellsrevealed that a SNARE assembly-inhibiting antibodyand clostridial neurotoxins reduce the initial fast com-ponent of the EXOCYTIC BURST, indicating that even themost ready-to-be-released vesicles have not bypassedthe SNARE assembly step49,57. These results obtainedfrom physiologically relevant systems are consistentwith those from the in vitro synthetic liposome fusionsystem, in which v- and t-SNAREs are separately recon-stituted into synthetic liposomes and fusion of the twoliposome populations is measured by either FLUORES-

CENCE RESONANCE ENERGY TRANSFER between lipids58 or con-tent mixing59. This system showed that SNAREs arenecessary and sufficient to mediate lipid and contentmixing58,59.

So, both in vitro and in vivo evidence indicate thatSNAREs might be involved in a late, if not the final, stepof membrane fusion. However, there have also beenreports to the contrary. In the YEAST VACUOLAR FUSION SYS-

TEM, it was reported that SNARE complexes could bedissociated and prevented from reassembly with ablocking antibody, and that this disruption of SNARE

EXOCYTIC BURST

Defined by Neher andcolleagues as the initial burst ofrelease occurring within a fewhundred milliseconds after thestimulus (in the chromaffin cellsystem), which is probably dueto exocytosis of secretorygranules that are in a release-ready state. It can be furtherresolved into two kineticallydistinct components.

FLUORESCENCE RESONANCE

ENERGY TRANSFER

Process of energy transferbetween two fluorophores. Canbe used to determine thedistance between twoattachment positions within amacromolecule or between twomolecules.

YEAST VACUOLAR FUSION

SYSTEM

In vitro fusion assay thatmeasures the homotypic fusionof vacuoles isolated from theyeast Saccharomyces cerevisiaeusing a colorimetric alkalinephosphatase assay.

SEA URCHIN EGG FUSION

SYSTEM

In vitro fusion assay thatmeasures the homotypic fusionof cortical vesicles isolated fromsea urchin eggs upon additionof calcium, by measuringturbidity (A

405).

ATPADP+Pi

Nucleation

Membranefusion

NSFNSF

NSF

α-SNAP

α-SNAP

α-SNAP

(Rab)Ca2+

Zipping

VAMP

SyntaxinSNAP-25

n-Sec1

n-Sec1

n-Sec1

n-Sec1

Figure 4 | Molecular model of vesicle exocytosis. Syntaxin is bound to n-Sec1 before formation of the core complex. Rabproteins might facilitate the dissociation of n-Sec1 from syntaxin, allowing subsequent binding (nucleation) between the threeneuronal SNAREs, syntaxin, SNAP-25 and VAMP (for simplicity, only one coil is drawn for SNAP-25). Ca2+ triggers the full zippingof the coiled-coil complex, which results in membrane fusion and release of vesicle contents. After the fusion event, recruitmentof α-SNAP and NSF from the cytoplasm and subsequent hydrolysis of ATP by NSF causes dissociation of the SNARE complex.Syntaxin, VAMP and SNAP-25 are then free for recycling and another round of exocytosis. (NSF; N-ethyl-maleimide-sensitivefusion protein; SNAP-25, 25 kDa synaptosome-associated protein; SNARE, soluble NSF attachment protein receptor, VAMP,vesicle-associated membrane protein.)



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B70,71, this indicates that the amino-terminal portion ofVAMP might be shielded in a protein complex beforethe arrival of the fusion trigger, implying the existence ofhalf-zipped SNARE complexes. In the adrenal chromaf-fin cell system, kinetic analysis of exocytosis using hightime resolution MEMBRANE CAPACITANCE MEASUREMENTS

revealed that SNAREs exist in a dynamic equilibriumbetween a loose and a tight form49. The two forms canbe distinguished by their different sensitivities to BOTU-

LINUM NEUROTOXIN A57 and antibodies that block SNAREcomplex formation49. In the artificial liposome system, itwas shown that prior incubation of v- and t-SNARE-containing liposomes resulted in liposome docking andan accelerated rate of fusion40,58. All of the above resultsindicate that inter-SNARE interactions might occurbefore the actual membrane fusion event. These interac-tions could be critical for establishing the readiness ofthe release machinery for fast fusion.

What prevents this partial complex from zippingfully? As Ca2+ is known to be the final trigger for manytrafficking steps4–7, one possibility is that the SNAREcomplex is prevented from fully assembling by a Ca2+-sensitive clamp. Synaptotagmin I, a synaptic protein thatbinds Ca2+, SNAREs and phospholipids has been impli-cated as the Ca2+ sensor in neuronal exocytosis72. It hasbeen proposed that synaptotagmin wraps around themembrane-proximal base of a preassembled trans-SNARE complex and triggers fast exocytosis through itsCa2+-dependent interactions with SNAREs and/or mem-branes73,74. It is uncertain, however, how this interactionwould prevent full SNARE assembly. It has also been sug-gested that either the interaction between synaptotagminand the carboxyl terminus of SNAP-25 (REFS 57,75) or theCa2+-induced oligomerization of synaptotagmin76 couldbe important for excitation–secretion coupling. Moreexperimental data are needed before we can build acoherent model of synaptotagmin and SNARE functionin synapses.

Mechanics of SNARE-mediated lipid fusionIt is energetically expensive to fuse two membranes in anaqueous environment, because the electrostatic repul-sive and hydration forces between the two membraneshas to be overcome77. In the current fusion model15,78,79

(FIG. 5), once the distance between the two bilayers is suf-ficiently reduced, HEMIFUSION occurs (FIG. 5c,d), followed bydistal leaflet membrane breakdown, resulting in theopening of a ‘fusion pore’ (FIG. 5e). Finally, the fusionpore expands, causing full content mixing and mem-brane relaxation (FIG. 5f). The first aqueous connectionbetween the lumen of the vesicle and the extracellularspace (or the lumen of another membrane compart-ment) — the fusion pore — was initially shown to existby both FREEZE–FRACTURE ELECTRON MICROSCOPY80 and PATCH-

CLAMP electrophysiological recordings81. The pore wasonce hypothesized to be initially proteinaceous, consist-ing of GAP-JUNCTION-like proteins that span two bilayers82.However, evidence has since indicated that the pore islikely to be purely lipidic, with proteins acting as anexternal scaffold15,78,79, as was initially suggested byFernandez and colleagues83. In such a model, directed

Do SNAREs assemble before final zipping? When do SNARE complexes form (BOX 3)? If zipping upthe SNAREs into a four-helix bundle drives the mem-brane fusion reaction then the SNARE complex shouldonly exist as a coiled-coil bundle (similar to the crystalstructure of an in vitro complex) during or after fusion.However, increasing evidence indicates that before thefusion signal arrives, a reversible or partially assembledtrans-SNARE complex (BOX 3) might exist. In crayfishneuromuscular junctions, it was found that the inhibi-tion of synaptic transmission by TETANUS TOXIN, but notbotulinum toxin B, requires prior nerve activity69. As theamino-terminal portion of VAMP is required for itsproteolysis by tetanus toxin, but not by botulinum toxin

Figure 5 | Model of SNARE-mediated lipid fusion. a | The two membranes are in the vicinityof each other but the SNAREs are not yet in contact. b | SNARE complexes start zipping fromthe amino-terminal end, which draws the two membranes further towards each other. c | Zipping proceeds, causing increased curvature and lateral tension of the membranes,exposing the bilayer interior. Spontaneous hemifusion occurs as the separation is sufficientlyreduced. d | The highly unfavourable void space at the membrane junction in (c) causes theestablishment of contacts between the distal membrane leaflets. e | The lateral tension in thetransbilayer contact area induces membrane breakdown, yielding a fusion pore. f | The fusionpore expands and the membrane relaxes. (SNARE, soluble NSF attachment protein receptor,where NSF stands for N-ethyl-maleimide-sensitive fusion protein.)

Box 3 | Formation of cis- and trans-SNARE complexes

In recent literature, there has been some confusion over the expression ‘SNAREcomplex formation’. Traditionally, SNAREs have been studied in vitro or indetergent-containing solutions, conditions under which only fully assembledcomplexes exist. However, in vivo, when the v- and t-SNARE proteins are anchored inmembrane lipids, they can be in cis (on the same membrane) or trans (on opposingmembranes) conformations. A SNARE complex in the cis conformation probablyresembles the fully assembled in vitro complex, whereas SNAREs in a trans complexmight only loosely or partially interact because of the resistance posed by themembranes, and its structure and properties are probably very different to the invitro complex. For example, a trans complex is unlikely to be SDS resistant,clostridial neurotoxin resistant57 or thermostable, and it might not require α-SNAPand NSF for dissociation101, unlike its cis counterpart. However, this distinction wasnot made experimentally until recently, so the term ‘SNARE complex formation’might have been used ambiguously in many of the earlier functional studies. Someauthors used it to describe pre-fusion partial assembly of trans SNAREs, whereasothers used it to describe the irreversible zipping up of the complex leading to cis-complex formation concurrent with membrane fusion. Some of the controversy inthe field is therefore only semantic.

TETANUS TOXIN

Clostridial neurotoxin thatcleaves VAMP.

MEMBRANE CAPACITANCE

MEASUREMENTS

Patch-clamp technique thatallows indirect measurementsof single exocytic events. Thetechnique measures the increasein the capacitance (andtherefore surface) of the plasmamembrane that results fromfusion of exocytic vesicles withthe plasma membrane.



R E V I E W S

tion does not greatly affect membrane fusion in eithersynthetic liposomes or cracked PC12 cells16,40,45, but fastkinetic analysis of the fusion reaction was not possiblein either system. It remains possible that a ring ofSNARE complexes is generated by some other means,such as homomultimerization mediated by transmem-brane domains51 or interactions with other regulatorsthat can oligomerize, such as synaptotagmin76.Development of single molecule analyses might be nec-essary to advance our understanding in this respect.

Although SNAREs alone are sufficient to fuse syn-thetic liposomes of certain lipid compositions, it hasbeen suggested that, at least in some cases, SNARE com-plex formation might induce a hemifusion intermediatestate, after which SNAREs become dispensable15. If thiswas the case then there would be a second catalyst totrigger the fusion pore opening and expansion, causingthe distal lipid layer and content mixing. This secondcatalyst could be synaptotagmin15 or conceivably an as-yet-unknown channel-like molecule that can formfusion pores. Or is it possible that Ca2+, the final triggerfor many membrane transport steps, could actually bethe second catalyst? Divalent cations, such as Ca2+, cancause significant changes in membrane tension77,86. Theconcentration of Ca2+ that is sensed at the fusion site hasbeen estimated to range from 25 µM at presynaptic ter-minals87 to about one to two orders of magnitude lowerin endocrine exocytosis57,64 and intracellular membranefusion6. Whether this wide range of Ca2+ concentrationscan trigger similar molecular events at distinct mem-brane fusion steps remains unknown.

PerspectivesA progressively more coherent molecular model ofSNARE-mediated membrane fusion is emerging fromrecent mechanistic studies. However, many remainingmysteries need to be solved before SNARE proteins canbe used to create artificial fusion systems for drug deliv-ery or to perturb secretion of hormones or other sig-nalling molecules in vivo. In addition to further under-standing how SNARE proteins act on membrane lipidsto cause fusion, we need to comprehend some of thekey regulatory mechanisms of cellular fusion, includingCa2+ regulation and Rab-GTPase-mediated regulation.Do Ca2+ ions act directly on membrane lipids, or acti-vate Ca2+-sensing proteins such as synaptotagminand/or calmodulin? How does activated synaptotagminor calmodulin then trigger membrane fusion? Whichfactors are involved in the Rab-GTP-initiated signallingpathway that leads to formation of the SNARE com-plex? How is the binding of syntaxin to its chaperone,n-Sec1, regulated? Furthermore, many newly identifiedproteins need to be better integrated into the SNARE-only picture presented above. For example, dozens ofproteins, including complexin88, tomosyn89, Hrs-2 (REF.

90), snapin91, syntaphilin92 and Munc-13 (REF. 93), wereidentified as potential factors in vesicle exocytosis; how-ever, their precise functions in membrane transport arestill largely unknown. Specific disruption of these pro-teins in functional assays and model organisms will per-haps be required to explain their function. Although

movement of membranes by proteins under conditionsof high membrane tension and curvature is essential; itis this scaffolding that creates the force that allowshemifusion and lipid fusion pore opening and expan-sion to occur83.

SNARE proteins are excellent candidates for doingthis. Their unusual ability to form a trans complexmight direct the two membranes towards each otherand create marked curvature and tension in the mem-branes, thereby stabilizing the transition state (FIG. 5A–C).In the liposome fusion system, insertion of a linkerbetween the transmembrane domain and the coil-forming domains of the SNAREs decreased fusion effi-ciency with increasing linker length53, indicating thatthere might be a stringent functional requirement for acertain length of this connection. Furthermore, replac-ing the membrane anchors of syntaxin and VAMP withshort phospholipids that do not span the bilayer pre-vented lipid mixing54. SNAREs might therefore exert aforce through the linker to the membrane anchors byforming a core complex, generating inward and lateralmovement in both membranes. As it is the zipping of atrans complex in a parallel fashion that generates thisforce, it is no surprise that the high in vitro stability ofthe core complex is functionally relevant. Mutation ofthe conserved hydrophobic residues that are importantfor core complex formation results in decreased ther-mostability of core complexes, which parallels adecreased ability of SNAREs to function in exocytosis inPC12 cells45. In addition to their transmembranedomains, SNAREs might be equipped to affect mem-branes in several ways. For example, there are basicamino acids at the membrane-proximal end of the corecomplex, which are well positioned to affect negativelycharged membrane surfaces52. Furthermore, two tryp-tophan residues and a tyrosine residue at the carboxy-terminal end of VAMP’s coil domain have been suggest-ed to be involved in potential SNARE–lipid interactionsduring fusion52. Further structure–function analyses areneeded to substantiate the functional importance ofthese residues.

This mechanism of SNARE-mediated fusion hassome similarity to viral-protein-mediated membranefusion. In both cases, coiled-coil helical bundles are themain structural component of the fusion protein, and apronounced conformational change of the protein pro-motes fusion15,84. The viral fusion is proposed to use a‘jack-knife’ mechanism, in which bending of a helixbundle brings the two membrane anchors and the asso-ciated bilayers together85, whereas SNAREs are pro-posed to use the zipper mechanism described above toachieve the same effect.

How many SNARE complexes are needed for onefusion event? Intuitively, a ring of complexes seems tobe the most optimal to apply uniform force on themembrane lipids to create a fusion pore. However, sofar there has been no experimental evidence to indicatethat several SNARE complexes might be required toopen one fusion pore. Although the linker region ofSNAP-25 between the two coiled-coil domains causesmultimerization of core complexes in vitro21, its dele-

BOTULINUM NEUROTOXIN A

Clostridial neurotoxin thatcleaves the SNAP-25 carboxy-terminal coil.

HEMIFUSION

Transient membrane fusionintermediate in which only thetwo proximal leaflets of thebilayer mix.

FREEZE–FRACTURE ELECTRON

MICROSCOPY

A technique in whichmembrane samples are deepfrozen and then fractured withthe blade of a knife to reveal theinternal structure of themembrane.

PATCH CLAMP

Technique whereby a very smallelectrode tip is sealed onto apatch of cell membrane, therebymaking it possible to record theflow of current throughindividual ion channels orpores within the patch.

GAP JUNCTION

Communicating junction(permeant to molecules up to 1kDa) between adjacent cells,which is composed of 12connexin protein subunits, sixof which form a connexon orhemichannel contributed byeach of the coupled cells.



R E V I E W S

systems with different techniques. In the future, with allthat is learned from different systems, we will be able tograsp not only what is conserved, but also what isunique to each transport step.

functional assays facilitate mechanistic understanding,it is often difficult to disrupt a given molecule specifical-ly. Knockout studies can be highly informative, but theyleave us with the daunting task of extracting mechanis-tic information from a phenotype. Another difficultyfacing the field is the fact that the molecules of interestoften occupy transient states. However, it is to ouradvantage that membrane fusion is a conserved featureof all eukaryotic cells and so can be studied in diverse

Links

DATABASE LINKS NSF | STX1 | SNAP-25 | VAMP1 | α-SNAP | n-Sec1 | calmodulin | synaptotagmin I |complexin | tomosyn | snapin | syntaphilin | Munc-13

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AcknowledgmentsWe thank S. Scales for critically reading the manuscript and L.Gonzalez, S. Scales, B. Yang and R. Lin for the artwork in FIGS 1, 2and 4.


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