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1.
Zhen Zhang 《Biophysical journal》2010,98(11):2524-2534
A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca2+-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca2+-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca2+-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape. 相似文献
2.
Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca2+-triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological
manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively
affect defined late stages of exocytosis, particularly the Ca2+-triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle
fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally
dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine
and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of
polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion
kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature.
Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine
on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have
modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming
steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust
high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming,
and triggered fusion. 相似文献
3.
The Ca2+-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca2+-independent manner with a lipid partition coefficient of approximately 3.0 × 102 M− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca2+. Although the Ca2+-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca2+. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca2+, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca2+-binding loops of C2B insert into the bilayer. This Ca2+-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery. 相似文献
4.
Che-Wei Chang Chung-Wei Chiang Jon D. Gaffaney Edwin R. Chapman Meyer B. Jackson 《The Journal of biological chemistry》2016,291(6):2848-2857
SNARE proteins catalyze many forms of biological membrane fusion, including Ca2+-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca2+-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca2+-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion. 相似文献
5.
Ca2+-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific
preparation to study the mechanisms by which Ca2+ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins
have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide
(IA). IA was found to enhance the kinetics and Ca2+ sensitivity of both CV-plasma membrane and CV–CV fusion. If Sr2+, a weak Ca2+ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca2+, suggesting that IA acts at the Ca2+-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol
sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with
fusion competency. 相似文献
6.
Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. 2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. 31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from 31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and 31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843. 相似文献
7.
Chunliang Xie Jianglin Li Tianyao Guo Yizhong Yan Cheng Tang Ying Wang Ping Chen Xianchun Wang Songping Liang 《Biochemical and biophysical research communications》2014
Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca2+-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes. 相似文献
8.
Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid
mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal
membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition:
at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made
accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium
addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM)
was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca2+, Mg2+, Cd2+ and La3+ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to
be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited
vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely
restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle
is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely
lipidic and protein-mediated membrane fusion is discussed.
Received: 27 September 1999/Revised: 22 March 2000 相似文献
9.
神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白 总被引:1,自引:0,他引:1
近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用. 相似文献
10.
Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca2+ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca2+-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and 2H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the 2H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of − 0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases. 相似文献
11.
Matts Nylund 《生物化学与生物物理学报:生物膜》2005,1669(2):87-94
The mammalian glycolipid transfer protein, GLTP, catalyzes the transfer in vitro of glycolipids between membranes. In this study we have examined on one hand the effect of the variations in the donor vesicle composition and on the other hand the effects of variations in the acceptor vesicle composition on the GLTP-catalyzed transfer kinetics of galactosylceramide between bilayer vesicles. For this purpose a resonance energy transfer assay was used, the energy donor being anthrylvinyl-galactosylceramide and the energy acceptor DiO-C16. First, we show that the transfer of anthrylvinyl-galactosylceramide from palmitoyl-oleoyl-phosphatidylcholine donor vesicles was faster than from dipalmitoyl-phosphatidylcholine vesicles, and that there is no transfer from palmitoyl-sphingomyelin vesicles regardless of the cholesterol amount. In this setup the acceptor vesicles were always 100% palmitoyl-oleoyl-phosphatidylcholine. We also showed that the transfer in general is faster from small highly curved vesicles compared to that from larger vesicles. Secondly, by varying the acceptor vesicle composition we showed that the transfer is faster to mixtures of sphingomyelin and cholesterol compared to mixtures of phosphatidylcholines and cholesterol. Based on these experiments we conclude that the GLTP mediated transfer of anthrylvinyl-galactosylceramide is sensitive to the matrix lipid composition and membrane bending. We postulate that a tightly packed membrane environment is most effective in preventing GLTP from accessing its substrates, and cholesterol is not required to protect the glycosphingolipid in the membrane from being transferred by GLTP. On the other hand GLTP can more easily transfer glycolipids to ‘lipid raft’ like membranes, suggesting that the protein could be involved in raft assembly. 相似文献
12.
Kuvichkin VV 《The Journal of membrane biology》2011,241(3):109-116
The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion
and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg2+, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced
fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered.
After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical
small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed.
ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded
by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles
with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out
the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins
begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic
energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered:
pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear
pore clusters. 相似文献
13.
The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca2+-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca2+-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca2+-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes. 相似文献
14.
JiangLi Li Yang Xiao Wei Zhou ZhengXing Wu RongYing Zhang Tao Xu 《中国科学:生命科学英文版》2009,52(12):1156-1163
Synaptotagmin VII (Syt VII), which has a higher Ca2+ affinity and slower disassembly kinetics with lipid than Syt I and Syt IX, was regarded as being uninvolved in synaptic vesicle (SV) exocytosis but instead possibly as a calcium sensor for the slower kinetic phase of dense core vesicles (DCVs) release. By using high temporal resolution capacitance and amperometry measurements, it was demonstrated that the knockdown of endogenous Syt VII attenuated the fusion of DCV with the plasma membrane, reduced the amplitude of the exocytotic burst of the Ca2+-triggered DCV release without affecting the slope of the sustained component, and blocked the fusion pore expansion. This suggests that Syt VII is the Ca2+ sensor of DCV fusion machinery and is an essential factor for the establishment and maintenance of the pool size of releasable DCVs in PC12 cells. 相似文献
15.
The function of synaptotagmin as a Ca2+ sensor in neurotransmitter release involves Ca2+-dependent phospholipid binding to its two C2 domains, but this activity alone does not explain why Ca2+ binding to the C2B domain is more critical for release than Ca2+ binding to the C2A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca2+ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca2+ binding to the C2B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C2B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release. 相似文献
16.
The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition. 总被引:8,自引:1,他引:7
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To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates. 相似文献
17.
Amritlal Mandal Sudip DasTapati Chakraborti Pulak KarBiswarup Ghosh Sajal Chakraborti 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
The properties of Ca2+-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C12E8) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than solubilization with C12E8 or Triton X-100. DHPC was determined to be superior to C12E8; while that the C12E8 was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca2+-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C12E8 and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca2+ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C12E8 and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca2+-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C12E8 and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca2+ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca2+-ATPase retained more organized and native secondary conformation compared to C12E8 and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C12E8 and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme. 相似文献
18.
Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (Lo) and liquid-disordered (Ld) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with Ld markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by Lo marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM1 enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM1/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘Lo/Ld’ boundary line tension arising from the coalescence of smaller Ld-like domains makes it energetically favourable for Ld-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events. 相似文献
19.
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. 相似文献
20.
The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using 2H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (ld) and liquid ordered (lo) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of ld and lo phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the ldand lo phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-Tm and one low-Tm lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid. 相似文献