Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.     

Diacylglycerol effects on phosphatidylinositol-specific phospholipase C activity and vesicle fusion

Diacylglycerol increased the hydrolytic activity of phosphatidylinositol-specific phospholipase C on large unilamellar vesicles containing 5-40% phosphatidylinositol. Moreover, diacylglycerol increased the rate and extent of vesicle fusion (contents mixing) induced by the enzyme. Kinetic studies of intervesicular lipid mixing revealed that fusion was limited by the frequency of contacts involving two diacylglycerol-rich domains.     

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR(2), a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR(2) is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR(2) when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR(2) cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR(2) on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR(2)-induced liposome aggregation, as long as Ca(2+) is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis相似文献   

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR2, a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR₂ is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR₂ when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR₂ cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR₂ on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR₂-induced liposome aggregation, as long as Ca²⁺ is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis < vesicle aggregation < total lipid mixing < inner lipid mixing < contents mixing. Concomitant measurements of the threshold diacylglyceride + ceramide concentrations in the bilayer show that late events, e.g. lipid mixing, require a higher concentration of PlcHR₂ products than early ones, e.g. aggregation. When the above results are examined in the context of the membrane effects of other phospholipid phosphocholine hydrolases it can be concluded that aggregation is necessary, but not sufficient for membrane fusion to occur, that diacylglycerol is far more fusogenic than ceramide, and that vesicle membrane permeabilization occurs independently from vesicle fusion.     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Liposome fusion catalytically induced by phospholipase C

Large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (50:25:25 mole ratio) were treated with phospholipase C. The early stages of phospholipid cleavage are accompanied by mixing of bilayer lipids (monitored by dequenching of octadecylrhodamine fluorescence) and leakage-free mixing of vesicle contents [measured by using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX)]. These results are interpreted in terms of vesicle fusion induced by the catalytic activity of phospholipase C. The use of sonicated unilamellar vesicles decreases the lag time, but does not modify the amplitude, of the fusion process. The presence of both phosphatidylethanolamine and cholesterol appears to be essential for measurable fusion effects to occur with low levels of phospholipid hydrolysis. Optimal fusion rates are observed with about 10-20 enzyme molecules per large unilamellar vesicle. This system of catalytically induced liposome fusion may be of relevance for the interpretation of physiological membrane fusion processes.     

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid Geometry and Bilayer Curvature Modulate LC3/GABARAP-Mediated Model Autophagosomal Elongation

Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here, we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologs (LC3, GABARAP, and GATE-16) to mediate membrane fusion. We found that both enzymatically and chemically lipidated forms of GATE-16 and GABARAP proteins promote extensive membrane tethering and fusion, whereas lipidated LC3 does so to a much lesser extent. Moreover, we characterize the GATE-16/GABARAP-mediated membrane fusion as a phenomenon of full membrane fusion, independently demonstrating vesicle aggregation, intervesicular lipid mixing, and intervesicular mixing of aqueous content, in the absence of vesicular content leakage. Multiple fusion events give rise to large vesicles, as seen by cryo-electron microscopy observations. We also show that both vesicle diameter and selected curvature-inducing lipids (cardiolipin, diacylglycerol, and lyso-phosphatidylcholine) can modulate the fusion process, smaller vesicle diameters and negative intrinsic curvature lipids (cardiolipin, diacylglycerol) facilitating fusion. These results strongly support the hypothesis of a highly bent structural fusion intermediate (stalk) during AP biogenesis and add to the growing body of evidence that identifies lipids as important regulators of autophagy.     

Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion

Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the "honeycomb structure") that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure ("stalk") would act as a structural intermediate in membrane fusion. Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.     

1,2-Dioleoylglycerol promotes calcium-induced fusion in phospholipid vesicles.

The effect of 1,2-dioleoyglycerol (1,2-DOG) on the promotion of Ca(2+)-induced fusion of phosphatidylserine/phosphatidylcholine (PS/PC) vesicles was studied. 1,2-DOG is able to induce the mixing of membrane lipids at concentrations of 10 mol% without mixing of vesicular contents. At concentrations of 20 mol% or higher, 1,2-DOG promotes fusion, lipid and content mixing, of LUV composed of an equimolar mixture of PS and PC, which otherwise are unable to fuse in the presence of Ca2+. Fusion was demonstrated by fluorescence assays monitoring mixing of aqueous vesicular contents and mixing of membrane lipids. Studies by Fourier transform infrared spectroscopy provided evidence for a fusion mechanism different to that of Ca(2+)-induced fusion of pure PS vesicles. Final equilibrium structures were characterized by 31P-NMR and freeze-fracture electron microscopy. Ca(2+)-induced fusion of 1,2-DOG containing vesicles is accompanied by the formation of isotropic structures which are shown to correspond to structures with lipidic particle morphology. The possible fusion mechanisms and implications are discussed.     

Fusion and phase separation monitored by lifetime changes of a fluorescent phospholipid probe

The sensitivity of the fluorescence lifetime of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]- 3-sn-phosphatidylcholine (DPHpPC) to its local concentration in lipid bilayers was used to monitor both lipid mixing and phase separation occurring during membrane vesicle fusion. Vesicles containing 2 mol % DPHpPC were mixed with a 10-fold excess of vesicles devoid of probe. Upon addition of a fusogen, mixing of bilayer lipids associated with fusion was followed as an increase in the fluorescence lifetime of DPHpPC. Ca2+-induced fusion of phosphatidylserine vesicles served to test the method and was shown to have an exponential half-time of 7 s. Phase separation (between the phosphatidylserine head groups of bulk lipid and the phosphatidylcholine head groups of the probe) was monitored by DPHpPC under the same conditions used to follow lipid mixing due to fusion. Phase separation was not significant until 10 min after Ca2+ addition and was completely reversible by disodium ethylenediaminetetraacetate addition. Vesicle aggregation induced by Ca2+ addition to mixed phosphatidylserine/phosphatidylcholine vesicles did not alter the DPHpPC lifetime, indicating that close association of vesicles did not promote intervesicular exchange of the probe. In addition, we have investigated the effects of CA2+ on the fluorescence properties of this probe and of the head-group-labeled fluorescent probes N-(4-nitro-2,1,3-benzoxadiazolyl)phosphatidylethanolamine and N-(lissamine Rhodamine B sulfonyl)dioleoyl-phosphatidylethanolamine, which are used in the fluorescence energy transfer assay of Struck et al.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.     

Ca2+-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.     

Ca-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.     

Interactions between human defensins and lipid bilayers: evidence for formation of multimeric pores.

Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions. Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the POPG:POPC ratio. Vesicles (300 microM POPG) readily aggregated at HNP-2 concentrations above 1 microM, but no mixing of vesicle contents could be detected for concentrations as high as 2 microM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 microM POPG vesicles, 50% of entrapped solute is released by 0.7 microM HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (approximately 400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.     

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

The rate of fusion of phospholipid vesicles and the role of bilayer curvature

Kinetics of Ca²⁺-induced fusion of phosphatidylserine vesicles is studiied for lipid concentrations varying from 1 μM to 100 μM. Fusion is monitored by mixing of aqueous vesicle contents and by explicitly accounting for leakage. The analysis provides separately rates of aggregation and fusion. The rate of fusion per se decreases steeply with vesicle size.     

Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.     

Roles of curvature and hydrophobic interstice energy in fusion: studies of lipid perturbant effects

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.     

Phospholipase C and sphingomyelinase activities of the Clostridium perfringens α-toxin

α-Toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. α-Toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of α-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5-7 range, in agreement with the proposed role of α-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.