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J. Biol. Chem., Vol. 281, Issue 40, 29597-29605, October 6, 2006

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Lipidic Antagonists to SNARE-mediated Fusion^*

From the Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, New York, New York 10032

Received for publication, February 24, 2006 , and in revised form, August 2, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Each membrane fusion event culminates in an organized assault on membrane integrity, rupturing two apposed bilayer surfaces in the process of fusing their lipid leaflets and mixing their respective contents. Not surprisingly, such an event is thermodynamically unfavorable, with the size of the energy barrier being in part a function of the intermediate structures sampled by the two membranes during the fusion event. The most successful model of these intermediate structures is the "stalk" model of membrane fusion (1–3) (see Fig. 1A), which predicts interaction of the apposed bilayers, followed by a transient hemi-fused state (the stalk), where the outer monolayers have merged, whereas the inner monolayers remain separated. Rupture of the stalk allows the formation of a pore (possibly with a "prepore" intermediate) that subsequently expands to complete the fusion of the bilayers.

In the course of stalk formation, interacting membranes must pass through transient intermediates that are highly curved. In a curved membrane the surface area to volume demands of the lipids on either side of the membrane are different. Manipulation of the lipidic composition of one leaflet of the membrane(s) can enhance or prevent certain types of curvature. Cone and inverted cone-shaped lipids have been used in a large number of pure lipid and viral-mediated fusion studies to introduce specific blocks or bursts to membrane bending and to allow an examination of the early stages of membrane fusion (4, 5) (see Fig. 1B).

The major proteins involved in overcoming the energetic barriers of membrane fusion are known for a wide variety of biological processes, most notably for enveloped viruses and intracellular trafficking. However, the molecular mechanisms employed to manipulate the lipid surfaces, the intermediate states sampled by both proteins and lipids, and the precise molecular organization of the fusion machinery remain largely unknown.

SNARE³ proteins are uniquely suited to biophysical characterization of the fusion event. They have a relatively simple architecture consisting of a transmembrane region, a SNARE domain, and at most one other presumably regulatory domain. This simplicity coupled with available in vitro fusion assays (6–8) has allowed a thorough characterization of the regions/residues that are essential for fusion. Furthermore, the machinery necessary for fusion is segregated into two membranes, which allows for the individual manipulation of the fusion components. The binding and zippering of coiled-coil domains derived from SNAREs on apposing membranes, SNAREpin assembly, provides the energy necessary to drive membrane fusion. This assembly process also includes at least one partially assembled intermediate in which the most membrane distal portions of the coiled-coil interact but zippering is incomplete (9–11). The extent of assembly and the degree of reversibility for this, or these, intermediates remains unknown.

We have previously used peptides derived from the SNAREs to isolate and describe an intermediate assembly stage during the neuronal SNARE-mediated fusion of reconstituted liposomes (9). Here we combine the use of SNARE-targeted fusion inhibitors and a battery of cone and inverted cone-shaped lipids to characterize the extent of complex formation in the SNAREpin when lipid membrane curvature becomes limiting.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Acyl-CoAs and oleic acid were purchased as powder from Sigma. Myristoyl-CoA displayed lot-dependent activities, including one preparation that activated fusion (lot number 22K70301); therefore only the several lots that inhibited fusion were used in this manuscript. Phospholipids were purchased in CHCl₃ from Avanti. Peptides derived from the membrane-proximal portion of the SNARE domain on VAMP2 were synthesized by the Microchemistry Core Facility of the Memorial Sloan-Kettering Cancer Center. The VAMP2-specific antibody 69.1 was purchased from synaptic systems.

Protein and Liposome Preparation—An N-terminally deleted syntaxin 1A, in which the first 150 amino acids were removed (comprising the whole of the N-terminal regulatory domain) was co-expressed with His₆-SNAP-25 and purified as described (6). t-SNARE complex containing His₆-SNAP-25 and syntaxin 1A lacking the transmembrane domain (Tsol), VAMP2-His₆, and His₆-VAMP2 lacking the transmembrane domain (Vsol) were each expressed and purified as described (9).

Fusion proteins of SNAREs with N-terminal fluorescent protein tags were generated as follows. ECFP-VAMP2 was cloned into the NcoI-XhoI sites on pET28a to form pYDQ1, which codes for a protein with a C-terminal His₆ tag. The coding sequence begins with the first methionine of enhanced cyan fluorescent protein (ECFP) and contains the entire sequence of ECFP, followed by an 11-amino acid linker (SGGSGGSGGEF), and the mVAMP2 sequence beginning with the first serine. EYFP-SNAP25 was cloned into the XhoI site of pET14b to form pYDQ3 and code for a protein with an N-terminal His₆ tag. The coding sequence includes all of enhanced yellow fluorescent protein (EYFP) followed by an 11-amino acid linker (SGGSGGSGGEF) and the mSNAP-25 sequence beginning with the first alanine. The EYFP also contains a Q69M mutation, generating the more photostable variant known as citrine (12). For protein expression of the fluorescent t-SNARE complex, a plasmid encoding full-length syntaxin 1A (pTW20) was co-transfected with pYDQ3 and purified via the His₆ tag on EYFP-SNAP25.

Small unilamellar proteoliposomes were prepared essentially as described (8), except that the liposomes contain a lower protein/lipid ratio. The syntaxin 1A/SNAP-25 liposomes were prepared at 1 t-SNARE/800 lipids, closer to surface densities that give rise to fusogenic supported bilayers (6), and the VAMP2 liposomes were prepared at 1 protein/100 lipids. An alternative expression of protein reconstitution is protein per liposome. We typically avoid this metric because it assumes a vesicle population having a homogeneous size. In our experience, detergent-dilution/dialysis liposomes are heterogeneous in size, with an average diameter of 35–40 nm (Ref. 13 and data not shown) but can include some liposomes greater than 100 nm in diameter. Furthermore, surface density rather than protein per liposome is likely the more relevant metric for considering physiological t-SNARE function, in which the protein is localized to target membranes rather than vesicles and is perhaps concentrated into rafts. However, to simplify comparisons across the literature, we have calculated an approximate protein/liposome; assuming each lipid headgroup occupies 65 Ų, a typical 35-nm liposome will have 9000 lipids and 11 t-SNAREs or 90 v-SNAREs. Botulinum B and tetanus toxin light chains were bacterially expressed from plasmids pBN13 and pOG-7, respectively, and purified via C-terminal His₆ tags.

Lipid Mixing Fusion Assay—The lipid mixing assay is a measure of fluorescence dequenching from NBD (nitrobenzoxadiazole)/rhodamine lipid pairs concentrated in the "donor" liposome population and diluted after fusion with "target" liposomes. The assay was run on a Spectramax plate reader essentially as described (9). t-SNARE liposomes were preincubated with buffer A (25 mM HEPES, pH 7.4, 100 mM KCl, 10% glycerol), Vsol, peptides, and/or lipidic molecules including the acyl-CoAs and lysolipids for 7 min at 37 °C prior to addition of the v-liposomes and initiation of the assay. Acyl-CoAs and oleic acid (OA) were prepared as aqueous suspensions in buffer A and added as <10% of the assay volume. Lyso-lipids were prepared as an emulsion in ethanol by bath sonication and added as <5% of the assay volume. Fatty acid-free BSA (Sigma) was prepared in buffer A immediately before use. The addition of material during an assay (buffer A, BSA, Vsol) sometimes caused a temporary decrease in the fusion rate caused by temperature fluctuations in the plate reader. In addition, when BSA was added to samples containing inhibitory lipidic molecules, an immediate, moderate decrease in fluorescence was routinely observed resulting from the sudden removal of the lipidic antagonists and consequent concentration (and requenching) of the fluorescently labeled lipids. Because the bulk of the volume in the assay derives from t-SNARE liposomes (45 µl versus 5 µl of v-SNARE and 5–10 µl of other compounds), we typically diluted the acyl-CoA into the t-liposome fraction. However, we also conducted numerous experiments in which the acyl-CoA was premixed with both the v- and the t-liposomes and observed identical results.

Acyl-CoAs inhibit fusion by wild-type syntaxin 1A (data not shown) just as effectively as fusion by syntaxin 1A lacking the auto-inhibitory N-terminal domain (14). We have used the N-terminal deletion variant throughout this manuscript, except where otherwise noted.

The results are presented as percentages of detergent signal, which is a normalization of the fusion-dependent fluorescence to the maximal fluorescent signal elicited with 0.4% dodecylmaltoside (Roche Applied Science) (8).

Fusion rates were calculated from the maximal slope of the assay rather than the initial slopes to avoid effects from the occasional temperature-dependent lag. Inhibitory and stimulated rates were normalized to control samples (samples without any added activating or inhibiting substances) after first subtracting the background drift observed in Vsol-inhibited samples to give: (experimental rate – background rate)/(control rate – background rate) x 100.

Fluorescence Resonance Energy Transfer (FRET) Assay—Liposome assays were performed as described for the lipid mixing assay above, except that the liposomes contained no fluorescent lipids. Instead, the proteins were directly modified with ECFP (VAMP2) or EYFP (SNAP-25). In both cases, the fluorescent proteins were added at the N termini of the SNARE proteins. When either of these proteins was tested in a lipid mixing assay, we could still observe an increase in fluorescence over time, suggesting that the N-terminal tags did not prevent fusion. The quantitative analysis of the fusion extent in these cases, however, was limited because of the high background fluorescence imposed by the fluorescent protein tags.

The samples were excited with 433-nm light at 37 °C in a PerkinElmer Life Sciences luminescence spectrometer (LS50B). Full emission spectra were recorded (for example, see Fig. 6A) after a 2-h incubation, from which the peak values of ECFP and EYFP emission were determined. Liposomes mixed in the presence of Vsol (to inhibit SNARE complex formation) elicit a spectrum that is identical to liposomes that are solubilized and proteolyzed (to liberate the fluorescent tag and thus eliminate any chance of FRET), indicating that the Vsol control is an accurate indicator of the No FRET base-line condition. FRET signal was measured as the change in the ratio of emission at 525 nm versus 480 nm upon protein complex formation. For time-dependent changes in FRET, the fluorescence intensity at these two wavelengths was recorded every minute in a Spectramax plate reader. The absolute intensity values differed somewhat between the Spectramax and PerkinElmer Life Sciences fluorimeters, such that the 525/480 ratio in any given experiment can only be compared with other experiments on the same machine.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Acyl-CoA-mediated Inhibition of SNARE Fusion—Inverted cone-shaped lipids (IC-lipids) are amphiphilic molecules having a large hydrophilic head group area as compared with the cross-sectional area of their hydrophobic tails. When applied to the outer leaflet of fusing membranes, they inhibit the type of hourglass curvature thought to be reminiscent of the stalk and fusion pore structure (Fig. 1, A and B). As such, they provide an avenue for blocking membrane fusion at or just before the formation of the stalk. To investigate intermediates in SNARE-mediated fusion, we screened a variety of IC-lipids for their ability to inhibit fusion in our reconstituted liposome assay.

All of the compounds that elicited some degree of inhibition are listed in Table 1. Lysophosphatidylethanolamine (lyso-PE) molecules having acyl-chains of either 14 or 16 carbons resulted in only a moderate level of inhibition. We could also consistently observe some inhibition by both lyso-PC and lysophosphatidylserine (lyso-PS); however, we avoided further experimentation with these molecules because of their greater propensity toward liposome solubilization at high concentration (15). In a similar study, Chen et al. (15) recently showed that lyso-PC could elicit 50% reduction in the rate of lipid mixing at concentrations between 100 and 330 µM. At higher concentrations, they observed an increase in fluorescence dequenching, signifying solubilization. We observed similar phenomena, with some compounds (like lyso-PC) exhibiting a plateau in inhibitory concentration. Indeed, a significant difficulty in characterizing IC-lipid behavior with our system is that the total lipid concentration is very high (1.5 mM), requiring high concentrations of inhibitor. Not surprisingly, detergent molecules like Tween 20 were also unusable. One class of molecules that worked well and did not result in liposome lysis is the family of fatty acyl-CoAs (Fig. 1C).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Membrane intermediates during fusion. A, a simplified cartoon of the stalk model of membrane fusion, in which the contacting (outer) leaflets of the two membranes fuse first to form a hemi-fused stalk structure. Rupture of the stalk leads to a fusion pore. In each of these intermediate structures, the membranes are believed to be highly curved. Eventually the pore expands, and the vesicle membrane completely collapses into the target membrane. B, magnification of the fusion pore (boxed in A). The formation of highly curved regions of the fusion pore or stalk structure is enhanced by the addition of different lipidic structures on the outer (cone-shaped, black triangles) or the inner (inverted cone-shaped, white triangles) leaflets of the membranes. C, structures of the inverted cone, palmitoyl-CoA, and the cone, oleic acid.

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Acyl-CoA is a concentration-dependent, reversible inhibitor of SNARE fusion. Acyl-CoAs were preincubated with t-SNARE liposomes for 7 min prior to the addition of v-liposomes and initiation of the assay. Acyl-CoAs (200 µM) inhibit SNARE-mediated lipid-mixing in an acyl chain length-dependent manner (A). % Detergent Signal indicates the fraction of fluorescent dequenching observed because fusion as compared with the maximum obtained in the presence of 0.4% dodecylmaltoside. B, the acyl-CoAs inhibit fusion in a concentration-dependent manner with half-maximal fusion occurring at 180 µM (bottom axis) or roughly 10 mol % total lipid (top axis). Each measurement is shown in triplicate, and the plot is representative of three experiments. C12 is lauryl-CoA, C14 is myristoyl-CoA, C16 is palmitoyl-CoA, and C18 is stearoyl-CoA.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Semi-assembled SNARE complexes accumulate during acyl-CoA inhibition. A, palmitoyl-CoA (200 µM) inhibition is reversed by the addition of BSA (190 µM). Palmitoyl-CoA was present at start of experiment (squares); BSA was added at 62 min (filled symbols). B, normalized fusion rates in the presence of 200 µM inhibitor (black bars) or inhibitor followed by 190 µM BSA addition (white bars) are plotted. Inset, fusion rates are normalized to maximal BSA-induced fusion rate in the presence of constant (200 µM) stearoyl-CoA and varying concentrations of BSA.

A hallmark of IC-lipid-mediated inhibition is reversibility by removal of the IC-lipids. BSA has as many as five independent sites for binding free fatty acids or fatty acyl moieties (21, 22); therefore we tested whether adding BSA during a fusion reaction could overcome the inhibition. The addition of BSA to a C16-inhibited fusion reaction caused an increase in the rate of fusion within 3 min of BSA application (Fig. 3A). After BSA addition, the rate of fusion in the inhibitor-containing sample was greater than at any point during the inhibitor-free control assay, suggesting that liposomes bound through partially assembled trans-SNARE complexes had accumulated in the presence of the inhibitor (Fig. 3). BSA had no effect on control (inhibitor-free) assays. Importantly, the acyl-CoAs are not activating an additional subpopulation of liposomes. The final level of fluorescence dequenching is the same in samples with and without IC-lipids, suggesting that the same numbers of liposomes proceed through fusion.

For convenience, most of our experiments were conducted by adding acyl-CoA to liposomes immediately before running the fusion assay. To establish that acyl-CoA was working within the plane of the membrane (rather than from an excess in solution), we preincubated liposomes with acyl-CoA and then refloated those liposomes over a nycodenz gradient to remove free IC-lipid. These treated liposomes were much less fusogenic than untreated and rapidly fused after BSA treatment, indicating that the acyl-CoA is absorbed into and active upon the membrane surface (data not shown).

The recovery rate is dependent upon the incubation time of the inhibitor lipid prior to BSA addition. For stearoyl-CoA-inhibited reactions, this rate of recovery continued to increase up to 45 min, indicating that new complexes continue to form over that time period (data not shown). The concentration of BSA needed to elicit 80% of the maximal recovery from 200 µM C16 was 72 µM (Fig. 3B, inset); therefore each fatty acid-free BSA was likely binding two or three molecules of acyl-CoA. The burst of fusion following BSA addition lasted only 10 min, after which the rate of fusion matched the control sample. Thus, all accumulated trans-SNARE-bound liposomes likely completed fusion within 10 min. From these collected results, we can conclude that a rate-limiting step for SNARE-mediated fusion of liposomes involves formation of the initial trans-SNARE complex, possibly including the organization of a higher order structure, prior to the development of a highly curved membrane surface.

Accumulated trans-SNARE Complexes Are Only Partially Assembled—trans-SNARE complexes form when the coiled-coil domains from SNAREs anchored in separate membranes come together. The precise organization of these complexes and the intermediate states sampled during their assembly are largely unknown. A popular hypothesis posits directed zippering of the coiled-coil domains of v- and t-SNAREs from their membrane distal N termini to their membrane proximal C termini to pull the membranes together. A variety of in vivo and in vitro results have been interpreted to support this model (9–11), but it remains controversial. For example, from single-molecule FRET experiments, a one-step assembly was proposed (23). Two similar electrophysiological studies of exocytosis from SNARE coiled-coil domain mutants also failed to produce a consensus model, with one group supporting the zippering hypothesis (24), whereas a second group favored a largely stochastic assembly process followed by a partial disassembly during pore-opening/expansion (25). Thus, the trans-SNARE state might be expected to have any of a number of SNARE surfaces exposed in our trapped intermediate. We next explored whether the lipid-inhibited complexes exhibited a degree of incomplete assembly (or partial disassembly) by utilizing a number of probes of SNARE-mediated fusion described previously.

There is only one other example of a stable fusion intermediate for reconstituted SNARE liposomes. When liposomes are incubated together at 4 °C, SNAREpin formation is kinetically trapped (Ref. 8 and Fig. 4A). Warming to 37 °C results in a burst of fusion as the preformed trans-SNARE complexes proceed through to fusion. These preformed complexes are largely resistant to subsequent addition of Vsol, indicating that they have assembled past the point of reversibility (because Vsol will occupy the same space as liposome-embedded VAMP2).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Vsol inhibits BSA-mediated recovery from acyl-CoA block. A, a previously observed Vsol-resistant intermediate (8) is recapitulated under our assay conditions. t- and v-liposomes were incubated overnight at 4 °C (squares) or premixed immediately before initiating assay (circles). The samples were challenged with buffer (filled symbols) or Vsol (open symbols). B and C, 190 µM BSA-mediated rescue from 200 µM stearoyl-CoA is blocked by Vsol addition (12 µM) either early (B) or late (C) in the assay. All of the experiments are representative of six or more assays.

View larger version (40K): [in this window] [in a new window]   FIGURE 5. Acyl-CoA blocked intermediates are resistant to toxin and peptide additions. A, VAMP2 primary structure with amino acid positions and tetanus- and botulinum toxin B-binding sites (hatched) and cleavage site (amino acid 76) indicated. SNARE domain encompasses the white area, polyproline rich encompasses the gray area, and transmembrane domain encompasses the black area. B, preincubation of either toxin with v-liposomes for 30 min at 37 °C completely abolished lipid-mixing. C, lipid mixing assay started in the presence of 200µM stearoyl-CoA followed by addition of toxin or buffer at 46 min and BSA (190 µM) at 98 min. D, lipid mixing assay started in the presence of 200 µM stearoyl-CoA followed by the addition of an inhibitory peptide (VnPep) derived from amino acids 29–56 of the N terminus of the VAMP2 SNARE domain (9) (10 µM; added at 32 min), followed by BSA (190 µM at 64 min). E, lipid mixing stimulated by C-terminal peptide (VcPep) derived from the VAMP2 SNARE domain amino acids 57–92 (9). All of the assays include 200 µM stearoyl-CoA. 190 µM BSA and 12 µM Vsol added where indicated.

We previously showed that peptides derived from the VAMP2 SNARE domain can be used to evaluate the extent of zippering (9). A peptide corresponding to the VAMP2 amino acids 29–56 (V29–56), making up roughly the N-terminal half of the VAMP2 SNARE domain, inhibits fusion prior to v-/t-SNARE complex formation. The C18-inhibited complexes are insensitive to V29–56 (Fig. 5D), consistent with a state of partial assembly involving the N-terminal halves of the SNARE domains.

A peptide derived from the C-terminal half of VAMP2 and comprising amino acids 57–92 (V57–92) accelerates fusion by inducing conformational changes over the membrane-proximal half of the t-SNARE complex, syntaxin 1A/SNAP-25 (9). We next asked whether stimulating these conformational changes could overcome the lipidic block. Fusion in the presence of V57–92 is still largely inhibited by C18 (Fig. 5E). Removal of the block, with BSA, again leads to a burst of fusion, and Vsol still abrogates the BSA-mediated relief. Thus, even with the stimulatory peptide present, the trans-SNARE complex cannot fully assemble if the membranes cannot bend.

FRET Indicates Accumulation of trans-SNARE Assemblies during Lipidic Block—The burst of fusion following BSA-mediated removal of the lipidic inhibitors indicates the accumulation of SNAREs at an intermediate state of assembly. These complexes are resistant to challenge with neurotoxins or the N-terminal VAMP2 peptide, suggesting that the epitopes for these reagents are buried in a multi-protein complex. Given the simplified composition of our reconstituted assay, these complexes must either be v-/t-SNARE assemblies (trans-SNAREs) or homomultimers of v- or t-SNAREs (most probably in the plane of the liposome membrane). A straightforward way to establish actual contact between v- and t-SNAREs is FRET.

We next generated v-SNAREs with fluorescent protein domains at the N terminus of VAMP2 (ECFP) and t-SNAREs composed of full-length syntaxin 1A and a fluorescently modified SNAP-25 (with an N-terminal EYFP) to monitor the time-dependent protein association via FRET. In the presence of the stimulatory V57–92 peptide, FRET between t- and v-liposomes is highly efficient (Fig. 6A). We chose to work under these conditions, which present the most stringent test of SNARE assembly during the lipidic block.

FRET between v- and t-liposomes develops with or without C18 (Fig. 6B), indicating the accumulation of trans-SNARE complexes in both cases. FRET develops quickly, faster than the lipid-mixing signal of comparable experiments, consistent with previous observations that interactions of the N termini of the SNAREs is fast in our liposome assay (9). The IC-lipid-induced block to lipid mixing must therefore be downstream of at least partial trans-SNARE assembly. Interestingly, after 1 h, the C18-containing samples exhibit slightly less total FRET (22%) than those without IC-lipid. The subsequent addition of BSA leads to a burst of FRET, eliminating most of this difference; however, the magnitude of this burst is much smaller than that observed for lipid-mixing, suggesting that most of the available SNAREs had already entered into trans-SNARE complexes during the block. What does the burst signify then? A likely possibility is that this signal arises from cis-SNARE complexes forming inside the liposomes, as they complete fusion, and as such, would represent a real time content mixing assay. Application of Vsol prior to BSA blocks the burst. Vsol does not cause a decrease in FRET, however, demonstrating that the preassembled SNARE complexes are not fully reversible. Rather, these complexes contain an exposed epitope, such that Vsol association prevents the final stages of assembly without completely displacing the membrane-embedded VAMP2.

Membrane Deformation Limits the Rate of SNARE-mediated Fusion—The accumulation of partially zippered SNARE intermediates and the burst of fusion following the removal of IC-lipids indicate that a rate-limiting step is the formation of these intermediates. The suggestion that these complexes are reversible in the presence of acyl-CoA indicates that completion of assembly is dependent upon the passage through a highly curved membrane intermediate. If assembly is directly affected by the membrane curvature, and assembly is rate-limiting, we expect that factors that enhance membrane curvature will enhance assembly and thus accelerate fusion. To test this prediction, we introduced cone-shaped lipid agonists to our fusion reaction.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. FRET of trans-SNARE complexes assembled between liposomes. v-liposomes are reconstituted with ECFP-VAMP2. t-liposomes are reconstituted with syntaxin 1A in complex with EYFP-SNAP-25. The fluorophores are positioned at the N termini of each protein, allowing them to come into close contact upon complex formation, providing a read-out of protein association even before lipid mixing has initiated. A, emission spectra of a mixture of v- and t-liposomes incubated in the presence of the V57–92 peptide to maximize protein assembly. Solid line, t-liposomes were premixed with Vsol to prevent FRET pairs from forming, before mixing with v-liposomes for 2 h at 37°C. Dashed line, t-liposomes were mixed with v-liposomes for 2 h before adding Vsol. In this case, FRET is apparent as the decrease in 480-nm emission and the increase in 525-nm emission. B, time-dependent development of FRET between liposomes. Liposome fusion assays were performed essentially as in Fig. 5E. The increase in FRET between liposomes is recorded as the change in the ratio of emission at 480 and 525 nm. 200 µM C14, 190 µM BSA, and Vsol added where indicated.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Fusion stimulation by the cone-shaped lipid OA. A, both lipid mixing rate and extent are increased by 70 or 200 µM OA. B, OA stimulation is concentration dependent (open circles) and completely suppressed by 182 µM BSA (filled squares). The addition of 200 µM stearoyl CoA (filled circles) suppresses the stimulation of OA at concentrations equal to or less than stearoyl CoA (top axis). In all of the experiments, oleic acid was added as an ethanolic emulsion (2 µl into a 55-µl assay), and control experiments received 2 µl of ethanol.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One caveat with IC-lipid studies is the possibility that the observed effects are not on membrane curvature but instead are a direct effect on the fusogenic protein itself (4). It is very difficult to rule out a direct lipid-protein interaction, but two lines of evidence suggest that what we are observing is a membrane curvature modification. First, the IC₅₀ concentrations across a wide range of potential IC-lipids are similar, indicating that mol % of total lipid is a critical driving force. Thus, when C18-acyl-CoA is added at 0.7 mol % of total lipid, there is almost no effect on fusion, even though the C18-acyl-CoA is in a 10-fold excess over t-SNARE. Second, when the acyl-CoA is restricted to the outer leaflet, the inhibitory activity can be overcome by including an equal concentration of a cone-shaped lipid.

The single best characterized attribute of the SNARE family is the ability to form highly stable four-helix coiled-coils, the cis-SNARE complex (27–29). The directed assembly of these complexes gives rise to membrane fusion in a variety of in vitro assays (6–8, 30), confirming the essential role of these proteins in fusion. We have now demonstrated that the assembly of SNAREs into an Vsol-resistant complex requires extensive membrane curvature and possibly fusion. Furthermore, the rate of fusion in our reconstituted system is limited by both the rate of SNAREpin assembly and by the rate at which membranes can be induced to curve, demonstrating that the two processes are tightly linked.

What might be the physiological significance of this intermediate? The readily releasable pool of synaptic vesicles is predocked to the synaptic membrane. When the SNARE disassembly capacity of NSF is reduced, more readily releasable vesicles accumulate (31), indicating that SNAREs are an essential determinant of the size of the pool. Furthermore, fusion from this pool of vesicles is completely blocked by the action of SNARE-proteolyzing neurotoxins and, in particular, those neurotoxins targeting the membrane-proximal region of the SNARE domain (10). Taken together, these results indicate a complex that is at least semi-assembled and is often modeled as being on the brink of fusion, perhaps having already restructured the membrane. Reversibility of this complex was first postulated by Scheller and co-workers (32), who suggest that in the "primed" state, SNAREs may be in a assembly/disassembly equilibrium, in anticipation of an appropriate trigger. The absence of toxin sensitivity in our assay suggests that SNAREs can zip up significantly beyond the predocked assembly stage of synaptic membranes and still present a binding site for Vsol. Our population FRET results do not lend any support to the notion of a fully reversible complex, but ultimately single-molecule FRET studies will be needed to capture transient uncouplings (33). By coupling final assembly of the SNAREpin to membrane fusion, the trans-SNARE complex can remain a malleable dynamic structure right up until the last moments at which the membrane becomes committed to lipid mixing.

In some instances fusion from the readily releasable pool does not lead to complete membrane mixture, culminating instead in a reversible fusion pore. These kiss and run events involve a transient fusion pore with a lifetime sufficient to release most or all of the vesicle contents. Subsequently, the vesicle "unfuses," pinching off to be available for reloading and another round. An intriguing mechanistic question surrounding these events remains: how the vesicle becomes "unSNAREd." Previous in vitro evidence has demonstrated that trans-SNARE pairs are not substrates for NSF recycling (34), and in any event the time scale of kiss and run appears to be too short for NSF binding and SNARE unfolding. Thus, an important avenue for further study is whether SNARE complex assemblies remain dynamic up to the point of fusion pore formation, with irreversible cis-SNARE pairs representing the end state of a fully dilated fusion pore.

The ability to preferentially isolate a single intermediate in membrane fusion will open the study of SNARE-mediated fusion to a variety of novel biophysical techniques. In addition, it will be possible to put the role of regulatory proteins, particularly those that activate the fusion complex and provide the calcium-trigger for regulated exocytosis, into context with regards to SNARE activation, SNARE assembly, and membrane curvature.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Present address: Genomics Core Facility, Dept of Molecular Biology, Memorial Sloan Kettering Cancer Center, 430 E. 67th St., New York, NY 10021.

¹ To whom correspondence should be addressed: Dept. of Physiology and Cellular Biophysics, Columbia University, 1150 Saint Nicholas Ave., New York, NY 10032. Tel.: 212-851-5569; Fax: 212-851-5570; E-mail: tm2176{at}columbia.edu.

³ The abbreviations used are: SNARE, soluble NSF attachment protein receptors; IC-lipid, inverted cone-shaped lipid; VAMP, vesicle-associated membrane protein; OA, oleic acid; BSA, bovine serum albumin; FRET, fluorescence resonance energy transfer; PC, phosphatidylcholine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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