SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation.

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.     

A role for soluble NSF attachment proteins (SNAPs) in regulated exocytosis in adrenal chromaffin cells.

Digitonin-permeabilized chromaffin cells secrete catecholamines by exocytosis in response to micromolar Ca2+ concentrations, but lose the ability to secrete in response to Ca2+ as the cells lose soluble proteins through the plasma membrane pores. Such secretory run-down can be retarded by cytosolic fractions, thus providing an assay for proteins potentially involved in the exocytotic process. We have used this assay to investigate the role of N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) in regulated exocytosis. Recombinant alpha- and gamma-SNAP stimulated Ca(2+)-dependent exocytosis, although recombinant NSF was ineffective, despite the fact that NSF and alpha-SNAP leak from the permeabilized cells with similar time courses. However, around one third of cellular NSF was found to be present in a non-cytosolic form and so it is possible that this is sufficient for exocytosis and that exogenous SNAPs stimulate the exocytotic mechanism by acting on the leakage-insensitive NSF. The stimulatory effect of alpha-SNAP displayed a biphasic dose-response curve and was maximal at 20 micrograms/ml. The effect of alpha-SNAP was Ca(2+)- and MgATP-dependent and was inhibited by N-ethylmaleimide and botulinum A neurotoxin, indicating a bona fide action on the exocytotic mechanism. Furthermore, Ca2+ concentrations which trigger catecholamine secretion acted to prevent the leakage of NSF and alpha-SNAP from permeabilized cells. These findings provide functional evidence for a role of SNAPs in regulated exocytosis in chromaffin cells.     

Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion

Much recent work has focussed on the role of membrane-bound components in fusion. We show here that p97 and NSF are sufficient to mediate rapid membrane fusion. Fractionation of cytosol revealed that p97 and its co-factor, p47, constitutes the major fusion activity. This was confirmed by depleting p97 from the cytosol, which resulted in an 80% decrease in fusion. Using purified protein, p97 or NSF was found to be sufficient to mediate rapid fusion in an ATP-dependent manner. A regulatory role was observed for their corresponding co-factors, p47 and alpha-SNAP. When present at a molar ratio half of that of the ATPase, both co-factors increased fusion activity significantly. Intriguingly, at this ratio the ATPase activity of the complex measured in solution was at its lowest, suggesting that the co-factor stabilizes the ATP state. The fusion event involved mixing of both leaflets of the opposing membranes and contents of liposomes. We conclude from these data that p97, NSF and perhaps other related ATPases catalyse rapid and complete fusion between lipid bilayers on opposing membranes. This highlights a new role for p97 and NSF and prompts a re-evaluation of current fusion models.     

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.     

Liposome-mediated transfer of integral membrane glycoproteins into the plasma membrane of cultured cells

Cultured mouse 3T3 cells treated with phosphatidylserine or phosphatidylserine/phosphatidylcholine (3: 7 mole ratio) liposomes containing ortho- and paramyxovirus envelope glycoproteins become susceptible to killing by virus-specific cytotoxic T lymphocytes indicating that the liposome-derived glycoproteins have been inserted into the cellular plasma membrane. Cells incubated with liposomes of similar lipid composition containing viral antigens plus a dinitrophenylated lipid hapten were killed by both virus- and hapten-specific T lymphocytes indicating that both protein and lipid components are inserted into the plasma membrane. We consider that assimilation of liposome-derived antigens into the plasma membrane results from fusion of liposomes with the plasma membrane. Cells incubated with phosphatidylcholine liposomes containing lipid haptens and viral glycoproteins were not killed by cytotoxic lymphocytes indicating that liposomes of this composition do not fuse with the plasma membrane. Liposome-derived paramyxovirus glycoproteins inserted into the plasma membrane retain their functional activity as shown by their ability to induce cell fusion. These experiments demonstrate the feasibility of using liposomes as carriers for introducing integral membrane (glyco)proteins into the plasma membrane of cultured cells and establish a new approach for studying the role of individual (glyco)proteins in the expression of specific cell surface properties.     

A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.     

Analysis of NSF mutants reveals residues involved in SNAP binding and ATPase stimulation

N-Ethylmaleimide-sensitive fusion protein (NSF) and its yeast orthologue, Sec18, are cytoplasmic AAA(+) ATPases required for most intracellular membrane fusion events. The primary function of NSF is thought to be the disassembly of cis-SNARE complexes, thus allowing trans-SNARE complex formation and subsequent membrane fusion. The importance of NSF/Sec18 in intracellular membrane traffic in vivo is highlighted by the inhibition of neurotransmission in Drosophila comatose (NSF) mutants and of constitutive secretion in yeast sec18 mutants. However, the underlying biochemical defects in these mutant proteins are largely unknown. Here, we identify the sec18-1 mutation as a G89D substitution in the N domain of Sec18p. This mutation results in an inhibition of the mutant protein's ability to bind to Sec17p (yeast alpha-SNAP). In contrast, engineering the comatose(st53)() mutation (S483L) into mammalian NSF (S491L) has no effect on alpha-SNAP binding. Instead, the stimulation of ATPase activity by alpha-SNAP required for wild-type NSF to disassemble SNARE complexes does not occur in the mutant NSF(st53) protein. This biochemical phenotype predicts a dominant negative effect, which was confirmed by engineering the st53 mutation into Sec18 (A505L), resulting in a dominant lethal phenotype in vivo. These findings suggest a biochemical basis for the block in membrane fusion observed in the mutant organisms. Furthermore, the mutants characterized here define key residues involved in two essential, but mechanistically distinct, biochemical functions of NSF: SNAP binding and SNAP-dependent ATPase stimulation.     

Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.     

Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion

BNIP1, a member of the BH3-only protein family, was first discovered as one of the proteins that are capable of interacting with the antiapoptotic adenovirus E1B 19-kDa protein. Here we disclose a totally unexpected finding that BNIP1 is a component of the complex comprising syntaxin 18, an endoplasmic reticulum (ER)-located soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE). Functional analysis revealed that BNIP1 participates in the formation of the ER network structure, but not in membrane trafficking between the ER and Golgi. Notably, a highly conserved leucine residue in the BH3 domain of BNIP1 plays an important role not only in the induction of apoptosis but also in the binding of alpha-SNAP, an adaptor that serves as a link between the chaperone ATPase NSF and SNAREs. This predicts that alpha-SNAP may suppress apoptosis by competing with antiapoptotic proteins for the BH3 domain of BNIP1. Indeed, overexpression of alpha-SNAP markedly delayed staurosporine-induced apoptosis. Our results shed light on possible crosstalk between apparently independent cellular events, apoptosis and ER membrane fusion.     

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) bind to a multi-SNAP receptor complex in Golgi membranes.

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) are required for the binding of N-ethylmaleimide-sensitive fusion protein (NSF) to Golgi membranes and are, therefore, required for intra-Golgi transport. We report the existence of distinct alpha/beta-SNAP and gamma-SNAP-binding sites in Golgi membranes that appear to be part of the same receptor complex. Cross-linking studies with alpha-SNAP demonstrate that an integral membrane protein of between 30-40 kDa is the alpha-SNAP binding component of the multi-SNAP receptor complex. These data suggest that SNAPs function by independently binding to a multi-SNAP membrane-receptor complex, thereby activating them to serve as adaptors for the targeting of NSF.     

Defining the SNARE complex binding surface of alpha-SNAP: implications for SNARE complex disassembly

N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein alpha-soluble NSF attachment protein (alpha-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of alpha-SNAP in this process, we used site-directed mutagenesis to identify residues in alpha-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine alpha-helices of alpha-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced alpha-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between alpha-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and alpha-SNAP work together to drive disassembly.     

The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.     

Mutual control of membrane fission and fusion proteins

Membrane fusion and fission are antagonistic reactions controlled by different proteins. Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles. Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles. Vps1p forms polymers that couple several t-SNAREs together. At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane. This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE. Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity. We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.     

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.     

Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.

The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.     

Effect of lipid composition on HVJ-mediated fusion of glycophorin liposomes to erythrocytes

We previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochem. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984]. In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied. Very little fusion occurred when glycophorin liposomes composed of dipalmitoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37 degrees C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.     

Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes

Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.