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1.
Protein machines and lipid bilayers both play central roles in cell membrane fusion, a process crucial to life. Recent results provide clues to how both components function in fusion. Recent observations suggest a common mechanism by which very different fusion machines (from lipid-enveloped viruses and synaptic vesicles) may function to produce compartment-joining pores. This mechanism presumes that fusion proteins act as machines that use stored conformational energy to assemble closely juxtaposed lipid bilayers, bend these to form fusion-competent structures, stabilize unfavorable lipid structures and destabilize a committed intermediate to drive fusion pore formation.  相似文献   

2.
The structural effects of the fusion peptide of feline leukemia virus (FeLV) on the lipid polymorphism of N-methylated dioleoylphosphatidylethanolamine were studied using a temperature ramp with sequential X-ray diffraction. This peptide, the hydrophobic amino-terminus of p15E, has been proven to be fusogenic and to promote the formation of highly curved, intermediate structures on the lamellar liquid-crystal to inverse hexagonal phase transition pathway. The FeLV peptide produces marked effects on the thermotropic mesomorphic behaviour of MeDOPE, a phospholipid with an intermediate spontaneous radius of curvature. The peptide is shown to reduce the lamellar repeat distance of the membrane prior to the onset of an inverted cubic phase. This suggests that membrane thinning may play a role in peptide-induced membrane fusion and strengthens the link between the fusion pathway and inverted cubic phase formation. The results of this study are interpreted in relation to models of the membrane fusion mechanism.  相似文献   

3.
Weinreb G  Lentz BR 《Biophysical journal》2007,92(11):4012-4029
We propose a model that accounts for the time courses of PEG-induced fusion of membrane vesicles of varying lipid compositions and sizes. The model assumes that fusion proceeds from an initial, aggregated vesicle state ((A) membrane contact) through two sequential intermediate states (I(1) and I(2)) and then on to a fusion pore state (FP). Using this model, we interpreted data on the fusion of seven different vesicle systems. We found that the initial aggregated state involved no lipid or content mixing but did produce leakage. The final state (FP) was not leaky. Lipid mixing normally dominated the first intermediate state (I(1)), but content mixing signal was also observed in this state for most systems. The second intermediate state (I(2)) exhibited both lipid and content mixing signals and leakage, and was sometimes the only leaky state. In some systems, the first and second intermediates were indistinguishable and converted directly to the FP state. Having also tested a parallel, two-intermediate model subject to different assumptions about the nature of the intermediates, we conclude that a sequential, two-intermediate model is the simplest model sufficient to describe PEG-mediated fusion in all vesicle systems studied. We conclude as well that a fusion intermediate "state" should not be thought of as a fixed structure (e.g., "stalk" or "transmembrane contact") of uniform properties. Rather, a fusion "state" describes an ensemble of similar structures that can have different mechanical properties. Thus, a "state" can have varying probabilities of having a given functional property such as content mixing, lipid mixing, or leakage. Our data show that the content mixing signal may occur through two processes, one correlated and one not correlated with leakage. Finally, we consider the implications of our results in terms of the "modified stalk" hypothesis for the mechanism of lipid pore formation. We conclude that our results not only support this hypothesis but also provide a means of analyzing fusion time courses so as to test it and gauge the mechanism of action of fusion proteins in the context of the lipidic hypothesis of fusion.  相似文献   

4.
Results of a kinetic model of thermotropic L alpha----HII phase transitions are used to predict the types and order-of-magnitude rates of interactions between unilamellar vesicles that can occur by intermediates in the L alpha----HII phase transition. These interactions are: outer monolayer lipid exchange between vesicles; vesicle leakage subsequent to aggregation; and (only in systems with ratios of L alpha and HII phase structural dimensions in a certain range or with unusually large bilayer lateral compressibilities) vesicle fusion with retention of contents. It was previously proposed that inverted micellar structures mediate membrane fusion. These inverted micellar structures are thought to form in all systems with such transitions. However, I show that membrane fusion probably occurs via structures that form from these inverted micellar intermediates, and that fusion should occur in only a sub-set of lipid systems that can adopt the HII phase. For single-component phosphatidylethanolamine (PE) systems with thermotropic L alpha----HII transitions, lipid exchange should be observed starting at temperatures several degrees below TH and at all higher temperatures, where TH is the L alpha----HII transition temperature. At temperatures above TH, the HII phase forms between apposed vesicles, and eventually ruptures them (leakage). In most single-component PE systems, fusion via L alpha----HII transition intermediates should not occur. This is the behavior observed by Bentz, Ellens, Lai, Szoka, et al. in PE vesicle systems. Fusion is likely to occur under circumstances in which multilamellar samples of lipid form the so-called "inverted cubic" or "isotropic" phase. This is as observed in the mono-methyl DOPE system (Ellens, H., J. Bentz, and F. C. Szoka. 1986. Fusion of phosphatidylethanolamine containing liposomes and the mechanism of the L alpha-HII phase transition. Biochemistry. In press.) In lipid systems with L alpha----HII transitions driven by cation binding (e.g., Ca2+-cardiolipin), fusion should be more frequent than in thermotropic systems.  相似文献   

5.
Membrane fusion proceeds via formation of intermediate nonbilayer structures. The stalk model of fusion intermediate is commonly recognized to account for the major phenomenology of the fusion process. However, in its current form, the stalk model poses a challenge. On one hand, it is able to describe qualitatively the modulation of the fusion reaction by the lipid composition of the membranes. On the other, it predicts very large values of the stalk energy, so that the related energy barrier for fusion cannot be overcome by membranes within a biologically reasonable span of time. We suggest a new structure for the fusion stalk, which resolves the energy crisis of the model. Our approach is based on a combined deformation of the stalk membrane including bending of the membrane surface and tilt of the hydrocarbon chains of lipid molecules. We demonstrate that the energy of the fusion stalk is a few times smaller than those predicted previously and the stalks are feasible in real systems. We account quantitatively for the experimental results on dependence of the fusion reaction on the lipid composition of different membrane monolayers. We analyze the dependence of the stalk energy on the distance between the fusing membranes and provide the experimentally testable predictions for the structural features of the stalk intermediates.  相似文献   

6.
Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.  相似文献   

7.
Thirty years ago, Klaus Arnold and others showed that the action of PEG in promoting cell–cell fusion was not due to such effects as surface absorption, cross-linking, solubilization, etc. Instead PEG acted simply by volume exclusion, resulting in an osmotic force driving membranes into close contact in a dehydrated region. This simple observation, based on a number of physical measurements and the use of PEG-based detergents that insert into membranes, spawned several important areas of research. One such area is the use of PEG to bring membranes into contact so that the role of different lipids and fusion proteins in membrane fusion can be examined in detail. We have summarized here insights into the fusion mechanism that have been obtained by this approach. This evidence indicates that fusion of model membranes (and probably cell membranes) occurs via severely bent lipidic structures formed at the point of sufficiently close contact between membranes of appropriate lipid composition. This line of research has also suggested that fusion proteins seem to catalyze fusion in part by reducing the free energy of hydrophobic interstices inherent to the lipidic fusion intermediate structures. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.  相似文献   

8.
May S 《Biophysical journal》2002,83(6):2969-2980
Fusion of lipid bilayers proceeds via a sequence of distinct structural transformations. Its early stage involves a localized, hemifused intermediate in which the proximal but not yet the distal monolayers are connected. Whereas the so-called stalk model most successfully accounts for the properties of the hemifused intermediate, there is still uncertainty about its microscopic structure and energy. We reanalyze fusion stalks using the theory of membrane elasticity. In our calculations, a short (cylindrical micelle-like) tether connects the two proximal monolayers of the hemifused membranes. The shape of the stalk and the length of the tether are calculated such as to minimize the overall free energy and to avoid the formation of voids within the hydrocarbon core. Our free energy expression is based on three internal degrees of freedom of a perturbed lipid layer: thickness, splay, and tilt deformations. Based on exactly the same model, we compare fusion stalks with and without the ability included to form sharp edges at the interfacial region between the hydrocarbon core and the polar environment. Requiring the interface to be smooth everywhere, our detailed calculations recover previous results: the stalk energies are far too high to account for the experimental observation of fusion intermediates. However, if we allow the interface to be nonsmooth, we find a remarkable reduction of the stalk free energy down to more realistic values. The corresponding structure of a nonsmooth stalk exhibits sharp edges at the transition regions between the bilayer and tether parts. In addition to that, a corner is formed at each of the two distal monolayers. We discuss the mechanism how membrane edges reduce the energy of fusion stalks.  相似文献   

9.
Haque ME  Lentz BR 《Biochemistry》2004,43(12):3507-3517
We have examined the effects of small amounts (1-4 mol %) of lipids of different molecular shapes, long chain lipids, and hydrocarbon on the kinetics of PEG-mediated fusion of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/sphingomyelin/cholesterol (DOPC/DOPE/SM/CH, 35:30:15:30) sonicated vesicles. The effects of these lipid perturbants were different for different steps in the fusion process and varied with the ratio of the cross-sectional areas of headgroup to acyl chain moieties. For lipids with a ratio <1 (negative intrinsic curvature), a decrease in this ratio led to a dramatic increase in the initial rate of vesicle contents mixing but left the initial rate of lipid mixing roughly unchanged. For lipids with ratios >1 (positive intrinsic curvature), the initial rates of both lipid and contents mixing decreased mildly with increasing ratio. In the context of the "stalk model" for fusion, lipid mixing reflects mainly formation of the initial fusion intermediate (stalk), while contents mixing reflects conversion of this intermediate either to a second intermediate or to a fusion pore. Results with positively curved lipids (ganglioside, GM1; lysophosphatidylcholine, LPCs) and negatively curved lipids (dioleoylglycerol, DOG, and 1,2-diphytanoyl-sn-glyvero-3-phosphatidylcholine, DPhPC) can be taken as supportive of the usual interpretation of the stalk model in terms of bending energy, but enhancement of fusion in the presence of long-chain phospholipids, hexadecane, as well as a mixture of GM1 plus hexadecane could not be explained by their curvature alone. We propose that the ability of a lipid perturbant to compensate for lipid packing mismatch, that is, to lower "void" energy, must be taken into account, along with intrinsic curvature, to explain the ability of lipid perturbants to promote pore formation.  相似文献   

10.
Energetics and dynamics of SNAREpin folding across lipid bilayers   总被引:3,自引:0,他引:3  
Membrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (approximately 2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k(B)T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k(B)T).  相似文献   

11.
We reported previously the effects of both osmotic and curvature stress on fusion between poly(ethylene glycol)-aggregated vesicles. In this article, we analyze the energetics of fusion of vesicles of different curvature, paying particular attention to the effects of osmotic stress on small, highly curved vesicles of 26 nm diameter, composed of lipids with negative intrinsic curvature. Our calculations show that high positive curvature of the outer monolayer "charges" these vesicles with excess bending energy, which then releases during stalk expansion (increase of the stalk radius, r(s)) and thus "drives" fusion. Calculations based on the known mechanical properties of lipid assemblies suggest that the free energy of "void" formation as well as membrane-bending free energy dominate the evolution of a stalk to an extended transmembrane contact. The free-energy profile of stalk expansion (free energy versus r(s)) clearly shows the presence of two metastable intermediates (intermediate 1 at r(s) approximately 0 - 1.0 nm and intermediate 2 at r(s) approximately 2.5 - 3.0 nm). Applying osmotic gradients of +/-5 atm, when assuming a fixed trans-bilayer lipid mass distribution, did not significantly change the free-energy profile. However, inclusion in the model of an additional degree of freedom, the ability of lipids to move into and out of the "void", made the free-energy profile strongly dependent on the osmotic gradient. Vesicle expansion increased the energy barrier between intermediates by approximately 4 kT and the absolute value of the barrier by approximately 7 kT, whereas compression decreased it by nearly the same extent. Since these calculations, which are based on the stalk hypothesis, correctly predict the effects of both membrane curvature and osmotic stress, they support the stalk hypothesis for the mechanism of membrane fusion and suggest that both forms of stress alter the final stages, rather than the initial step, of the fusion process, as previously suggested.  相似文献   

12.
The structural effects of in situ production of diacylglycerol by phospholipase C in pure lipid model membranes have been examined by freeze fracture electron microscopy. Phospholipase C-activity induces massive aggregation and fusion of large unilamellar lipid vesicles and leads to the formation of a 'sealed' lipid aggregate; the outer membrane of this aggregate appears to be continuous. In some areas lipid arranges into a honeycomb structure; this structure is probably a precursor of a discontinuous inverted (type II) cubic phase. Similarly, enzyme treatment of multilamellar vesicles leads to extensive membrane fusion and vesiculation. Thus morphological evidence is obtained showing the ability of phospholipase C to induce bilayer destabilization and fusion. It is speculated that phospholipase C-induced membrane fusion involves a type II fusion intermediate induced by diacylglycerol produced locally.  相似文献   

13.
We studied the mechanism of the lamellar-to-inverted hexagonal (L alpha/H[II]) phase transition, using time-resolved cryotransmission electron microscopy (TRC-TEM), 31P-NMR, and differential scanning calorimetry. The transition was initiated in dispersions of large unilamellar vesicles of dipalmitoleoyl phosphatidylethanolamine (DiPoPE). We present evidence that the transition proceeds in three steps. First, many small connections form between apposed membranes. Second, the connections aggregate within the planes of the bilayers, forming arrays with hexagonal order in some projections. Third, these quasihexagonal structures elongate into small domains of H(II) phase, acquiring lipid molecules by diffusion from contiguous bilayers. A previously proposed membrane fusion mechanism rationalizes these results. The modified stalk theory predicts that the L alpha/H(II) phase transition involves some of the same intermediate structures as membrane fusion. The small interbilayer connections observed via TRC-TEM are compatible with the structure of a critical intermediate in the modified stalk mechanism: the trans monolayer contact (TMC). The theory predicts that 1) TMCs should form starting at tens of degrees below TH; 2) when TMCs become sufficiently numerous, they should aggregate into transient arrays like the quasihexagonal arrays observed here by TRC-TEM; and 3) these quasihexagonal arrays can then elongate directly into H(II) phase domains. These predictions rationalize the principal features of our data, which are incompatible with the other transition mechanisms proposed to date. Thus these results support the modified stalk mechanism for both membrane fusion and the L alpha/H(II) phase transition. We also discuss some implications of the modified stalk theory for fusion in protein-containing systems. Specifically, we point out that recent data on the effects of hydrophobic peptides and viral fusion peptides on lipid phase behavior are consistent with an effect of the peptides on TMC stability.  相似文献   

14.
Siegel DP 《Biophysical journal》2008,95(11):5200-5215
The Gaussian curvature elastic energy contribution to the energy of membrane fusion intermediates has usually been neglected because the Gaussian curvature elastic modulus, κ, was unknown. It is now possible to measure κ for phospholipids that form bicontinuous inverted cubic (QII) phases. Here, it is shown that one can estimate κ for lipids that do not form QII phases by studying the phase behavior of lipid mixtures. The method is used to estimate κ for several lipid compositions in excess water. The values of κ are used to compute the curvature elastic energies of stalks and catenoidal fusion pores according to recent models. The Gaussian curvature elastic contribution is positive and similar in magnitude to the bending energy contribution: it increases the total curvature energy of all the fusion intermediates by 100 units of kBT or more. It is important to note that this contribution makes the predicted intermediate energies compatible with observed lipid phase behavior in excess water. An order-of-magnitude fusion rate equation is used to estimate whether the predicted stalk energies are consistent with the observed rates of stalk-mediated processes in pure lipid systems. The current theory predicts a stalk energy that is slightly too large, by ∼30 kBT, to rationalize the observed rates of stalk-mediated processes in phosphatidylethanolamine or N-monomethylated dioleoylphosphatidylethanolamine systems. Despite this discrepancy, the results show that models of fusion intermediate energy are accurate enough to make semiquantitative predictions about how proteins mediate biomembrane fusion. The same rate model shows that for proteins to drive biomembrane fusion at observed rates, they have to perform mediating functions corresponding to a reduction in the energy of a purely lipidic stalk by several tens of kBT. By binding particular peptide sequences to the monolayer surface, proteins could lower fusion intermediate energies by altering the elastic constants of the patches of lipid monolayer that form the stalk. Here, it is shown that if peptide binding changes κ or some other combinations of local elastic constants by only tens of percents, the stalk energy and the energy of catenoidal fusion pores would decrease by tens of kBT relative to the pure lipid value. This is comparable to the required mediating effect. The curvature energies of stalks and catenoidal fusion pores have almost the same dependence on monolayer elastic constants as the curvature energies of the rhombohedral and QII phases; respectively. The effects of isolated fusion-relevant peptides on the energies of these intermediates can be determined by studying the effects of the peptides on the stability of rhombohedral and QII phases.  相似文献   

15.
The widely accepted pathway of membrane fusion begins with the fusion stalk representing the initial intermediate of hemifusion. The lipid structures preceding hemifusion and their possible influence on fusion kinetics were not addressed. Here, we suggest the point-like protrusion as a prestalk fusion intermediate, which has energy lower than that of stalk and, therefore, does not limit the fusion rate. We demonstrate that by calculating the energy of the point-like protrusion, which depends on the lipid monolayer elastic parameters and the strength of the intermembrane hydration repulsion. The point-like protrusion completes the fusion-through-hemifusion model of membrane merger.  相似文献   

16.
Membrane fusion, essential to eukaryotic life, is broadly envisioned as a three-step process proceeding from contacting bilayers through two semistable, nonlamellar lipidic intermediate states to a fusion pore. Here, we introduced a new, to our knowledge, experimental approach to gain insight into the nature of the transition states between initial, intermediate, and final states. Recorded time courses of lipid-mixing, content-mixing, and content-leakage associated with fusion of 23 nm vesicles in the presence of poly(ethylene glycol) at multiple temperatures were fitted globally to a three-step sequential model to yield rate constants and thereby activation thermodynamics for each step of the process, as well as probabilities of occurrence of lipid-mixing, content-mixing, or content-leakage in each state. Experiments with membranes containing hexadecane, known to reduce interstice energy in nonlamellar structures, provided additional insight into the nature of fusion intermediates and transition states. The results support a hypothesis for the mechanism of stalk formation (step-1) that involves acyl chain protrusions into the interbilayer contact region, a hypothesis for a step-2 mechanism involving continuous interconversion of semistable nonlamellar intermediates, and a hypothesis for step-3 (pore formation) mechanism involving correlated movement of whole lipid molecules into hydrophobic spaces created by geometry mismatch between intermediate structures.  相似文献   

17.
Dynamics of fusion pores connecting membranes of different tensions   总被引:1,自引:0,他引:1       下载免费PDF全文
The energetics underlying the expansion of fusion pores connecting biological or lipid bilayer membranes is elucidated. The energetics necessary to deform membranes as the pore enlarges, in some combination with the action of the fusion proteins, must determine pore growth. The dynamics of pore growth is considered for the case of two homogeneous fusing membranes under different tensions. It is rigorously shown that pore growth can be quantitatively described by treating the pore as a quasiparticle that moves in a medium with a viscosity determined by that of the membranes. Motion is subject to tension, bending, and viscous forces. Pore dynamics and lipid flow through the pore were calculated using Lagrange's equations, with dissipation caused by intra- and intermonolayer friction. These calculations show that the energy barrier that restrains pore enlargement depends only on the sum of the tensions; a difference in tension between the fusing membranes is irrelevant. In contrast, lipid flux through the fusion pore depends on the tension difference but is independent of the sum. Thus pore growth is not affected by tension-driven lipid flux from one membrane to the other. The calculations of the present study explain how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. In a similar fashion, swelling of secretory granules after fusion in biological systems could promote pore enlargement during exocytosis. The calculations also show that pore expansion can be caused by pore lengthening; lengthening may be facilitated by fusion proteins.  相似文献   

18.
Li X  Schick M 《Biophysical journal》2001,80(4):1703-1711
The design of vesicles that become unstable at an easily tuned value of pH is of great interest for targeted drug delivery. We present a microscopic theory for two forms of such vesicles. A model of lipids introduced by us previously is applied to a system of ionizable anionic lipid and permanently charged cationic lipid. We calculate the pH at which the lamellar phase becomes unstable with respect to an inverted hexagonal one, a value that depends continuously on the system composition. Identifying this instability with that displayed by unilamellar vesicles undergoing fusion, we obtain very good agreement with the recent experimental data of Hafez, Ansell, and Cullis, (2000, Biophys. J. 79:1438-1446) on the pH at which fusion occurs versus vesicle composition. We explicate the mechanism in terms of the role of the counterions. This understanding suggests that a system of a neutral, nonlamellar-forming lipid stabilized by an anionic lipid would serve equally well for preparing tunable, pH-sensitive vesicles. Our calculations confirm this. Further, we show that both forms of vesicle have the desirable feature of exhibiting a regime in which the pH at instability is a rapidly varying function of the vesicle composition.  相似文献   

19.
We use continuum mechanics to calculate an entire least energy pathway of membrane fusion, from stalk formation, to pore creation, and through fusion pore enlargement. The model assumes that each structure in the pathway is axially symmetric. The static continuum stalk structure agrees quantitatively with experimental stalk architecture. Calculations show that in a stalk, the distal monolayer is stretched and the stored stretching energy is significantly less than the tilt energy of an unstretched distal monolayer. The string method is used to determine the energy of the transition barriers that separate intermediate states and the dynamics of two bilayers as they pass through them. Hemifusion requires a small amount of energy independently of lipid composition, while direct transition from a stalk to a fusion pore without a hemifusion intermediate is highly improbable. Hemifusion diaphragm expansion is spontaneous for distal monolayers containing at least two lipid components, given sufficiently negative diaphragm spontaneous curvature. Conversely, diaphragms formed from single-component distal monolayers do not expand without the continual injection of energy. We identify a diaphragm radius, below which central pore expansion is spontaneous. For larger diaphragms, prior studies have shown that pore expansion is not axisymmetric, and here our calculations supply an upper bound for the energy of the barrier against pore formation. The major energy-requiring deformations in the steps of fusion are: widening of a hydrophobic fissure in bilayers for stalk formation, splay within the expanding hemifusion diaphragm, and fissure widening initiating pore formation in a hemifusion diaphragm.  相似文献   

20.
Membrane fusion is fundamental to the life of eukaryotic cells. Cellular trafficking and compartmentalization, import of food stuffs and export of waste, inter-cellular communication, sexual reproduction, and cell division are all dependent on this basic process. Yet, little is known about the molecular mechanism(s) by which fusion occurs. It is known that fusing membranes must somehow be docked and brought into close contact. Specific proteins, many of which have been identified within the past decade, accomplish this. An electrical connection or 'fusion pore' is established between compartments surrounded by the fusing membranes. Three primary views of the mechanism of pore formation during secretory and viral fusion have been proposed within the past decade. In one view, a protein ring forms an initial transient connection that expands slowly by recruiting lipid so as to form a lipidic junction. In another view, the initial fusion pore consists of a protein-lipid complex that transforms slowly until the fusion proteins dissociate from the complex to form an irreversible lipidic pore. In a third view, the initial pore is a transient lipid pore that fluctuates between open and closed states before either expanding irreversibly or closing. Recent work has helped define the mechanism by which poly(ethylene glycol) (PEG) mediates fusion of highly curved model membranes composed only of synthetic phospholipids. PEG is a highly hydrated polymer that can bring vesicle membranes to near molecular contact by making water between them thermodynamically unfavourable. Disrupted packing in the contacting monolayers of these vesicle membranes is necessary to induce fusion. The time course and sequence of molecular events of the ensuing fusion process have also been defined. This sequence of events involves the formation of an initial, transient intermediate in which outer leaflet lipids have mixed and small transient pores join fusing compartments ('stalk'). The transient intermediate transforms in 1-3 min to a fusion-committed, second intermediate ('septum') that then 'pops' to form the fusion pore. Inner leaflet mixing, which is shown to be distinct from outer leaflet mixing, accompanies contents mixing that marks formation of the fusion pore. Both the sequence of events and the activation energies of these events correspond well to those observed in viral membrane fusion and secretory granule fusion. These results strongly support the contention that both viral and secretory fusion events occur by lipid molecule rearrangements that can be studied and defined through the use of PEG-mediated vesicle fusion as a model system. A possible mechanism by which fusion proteins might mediate this lipidic process is described.  相似文献   

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