共查询到20条相似文献,搜索用时 0 毫秒
1.
Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion. 相似文献
2.
Recent studies show that NSF, isolated over 15 years ago as a protein required for membrane fusion in vitro, can be reversibly inactivated by both S-nitrosylation and tyrosine phosphorylation. Different cell types use distinct post-translational modifications of NSF for localized regulation of membrane fusion. 相似文献
3.
4.
Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins. 相似文献
5.
Lipids in biological membrane fusion 总被引:8,自引:0,他引:8
The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions.Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion. 相似文献
6.
7.
8.
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
9.
Joshua Zimmerberg 《Bioscience reports》1987,7(4):251-268
Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion. 相似文献
10.
Sánchez-Magraner L Cortajarena AL Goñi FM Ostolaza H 《The Journal of biological chemistry》2006,281(9):5461-5467
Escherichia coli alpha-hemolysin (HlyA) is a protein exotoxin that binds and lyses eukaryotic cell and model membranes in the presence of calcium. Previous studies have been able to distinguish between reversible toxin binding to the membrane and irreversible insertion into the lipid matrix. Membrane lysis occurs as the combined effect of protein insertion plus a transient perturbation of the membrane bilayer structure. In the past, insertion and bilayer perturbation have not been experimentally dissected. This has now been achieved by studying HlyA penetration into lipid monolayers at the air-water interface, in which three-dimensional effects (of the kind required to break down the bilayer permeability barrier) cannot occur. The study of native HlyA, together with the nonlytic precursor pro-HlyA, and of different mutants demonstrates that although some nonlytic variants (e.g. pro-HlyA) exhibit very low levels of insertion, others (e.g. the nonlytic mutant HlyA H859N) insert even more strongly than the lytic wild type. These results show that insertion does not necessarily lead to membrane lysis, i.e. that insertion and lysis are not "coupled" phenomena. Millimolar levels of Ca(2+), which are essential for the lytic activity, cause an extra degree of insertion but only in the case of the lytic forms of HlyA. 相似文献
11.
Membrane fusion during spermiogenesis in Ascaris 总被引:4,自引:0,他引:4
12.
Deployment of membrane fusion protein domains during fusion 总被引:2,自引:0,他引:2
It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best-described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N- and C-termini of SNAP25 (sn25), together with the Ca(2+)binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA- mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N-termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the 'transition state' of the committed step of fusion. It is stabilized by a 'dam' of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion proteins SV5 F1 and HIV gp120/41, and for the intracellular SNARE fusion system. By comparing these domains, we have constructed a minimal set which appears to be adequate to explain how the conformational changes can produce a successful fusion event, i.e. communication of aqueous compartments. 相似文献
13.
Ralph J. Kessler Takashi Wakabayashi David E. Green 《Journal of bioenergetics and biomembranes》1974,6(5):205-220
The rod outer segment disk membrane of bovine retina has been isolated in a predominantly fused state. The physical and chemical properties of the membrane in the fused state are profoundly different from the corresponding properties of the same membrane in the unfused state. Exposure to light induces the transition of the disk membrane from the fused to the unfused state. Evidence is presented which suggests that the fusion-defusion cycle of the disk membrane is a primary event of photoexcitation and nerve stimulation. 相似文献
14.
The kinetics of lipid mixing during membrane aggregation and fusion was monitored by two assays employing resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and N-(lissamine Rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE). For the "probe mixing" assay, NBD-PE and Rh-PE were incorporated into separate populations of phospholipid vesicles. For the "probe dilution" assay, both probes were incorporated into one population of vesicles, and the assay monitored the dilution of the molecules into the membrane of unlabeled vesicles. The former assay was found to be very sensitive to aggregation, even when the internal aqueous contents of the vesicles did not intermix. Examples of this case were large unilamellar vesicles (LUV) composed of phosphatidylserine (PS) in the presence of Mg2+ and small unilamellar vesicles (SUV) composed of phosphatidylserine in the presence of high concentrations of Na+. No lipid mixing was detected in these cases by the probe dilution assay. Under conditions where membrane fusion (defined as the intermixing of aqueous contents with concomitant membrane mixing) was observed, such as LUV (PS) in the presence of Ca2+, the rate of probe mixing was faster than that of probe dilution, which in turn was faster than the rate of contents mixing. Two assays monitoring the intermixing of aqueous contents were also compared. The Tb/dipicolinic acid assay reported slower fusion rates than the 1-aminonaphthalene-3,6,8-trisulfonic acid/N,N'-p-xylylene-bis(pyridinium bromide) assay for PS LUV undergoing fusion in the presence of Ca2+. These observations point to the importance of utilizing contents mixing assays in conjunction with lipid mixing assays to obtain the rates of membrane destabilization and fusion.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol 总被引:2,自引:2,他引:0
Susan J. Quirk Quet Fah Ahkong Gaynor M. Botham Jan Vos Jack A. Lucy 《The Biochemical journal》1978,176(1):159-167
1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca2+ used, at least 80% of the cells fused after 30min at 37°C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in `ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca2+, although some cells fused when no exogenous Ca2+ was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of `ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1–2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca2+ into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed. 相似文献
16.
The mechanism of sperm-egg fusion in mammals is a research area that has greatly benefited from the use of gene deletion technology. Because fertilization is internal in mammals and the gametes (particularly the eggs) are sparse in number, in vitro studies have considerable limitations. Using gene deletions, a few cell surface proteins in both gametes have been identified as essential for gamete fusion. Ongoing studies are directed at analysis of the function of these proteins and the search for additional proteins that may be involved in this process. So far, no mammalian proteins have been found that also function in sperm-egg fusion of non-mammalian species or in other types of cell-cell fusion. 相似文献
17.
Intermediary structures during membrane fusion as observed by cryo-electron microscopy 总被引:2,自引:0,他引:2
Lipidic phases, containing 'lipidic particles' (dioleoylphosphatidylethanolamine/cholesterol/dioleoylphosphatidylcho lin e and cardiolipin/dimyristoylphosphatidylcholine in the presence of Ca2+) have been investigated by preparing thin films from a suspension of sonicated vesicles. These thin films were vitrified and observed 'directly' by cryo-electron microscopy in their hydrated form. The thin films show various fusion products and fusion intermediates such as lipidic particles. 相似文献
18.
Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed. 相似文献
19.
Membrane fusion in prokaryotes: bacteriophage phi 6 membrane fuses with the Pseudomonas syringae outer membrane. 总被引:5,自引:2,他引:3
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Protein-triggered membrane fusion in the prokaryotic system is described using the lipid-containing enveloped bacterial virus phi 6 and its host, the Gram-negative bacterium Pseudomonas syringae. Bacteriophage particles can be fused to form multiple particles where two or more nucleocapsids are surrounded by a single membrane vesicle with a volume proportional to the number of fused particles. For fusion to occur, a fusogenic protein is required in the membrane of the participating phage particles. Upon infection of the host cell, fusion of the viral membrane with the bacterial membrane takes place without leakage of the periplasmic enzyme alkaline phosphatase to the extracellular supernatant. There is a time-dependent mixing of fluorescent phage phospholipids with the bacterial membrane lipids between 5 and 20 min post-infection. The phage membrane proteins and phospholipids co-purify with the bacterial outer membrane of infected cells. The fusion is independent of divalent cations and pH, resembling Sendai virus fusion with the plasma membrane. This is the first targeted, protein-dependent fusion event described in prokaryotes. 相似文献
20.
Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components. This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals. 相似文献