首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Transmembrane topography and evolutionary conservation of synaptophysin   总被引:21,自引:0,他引:21  
Synaptophysin is the major integral membrane protein of small synaptic vesicles. Its primary structure deduced from rat and human complementary DNA sequences predicts that synaptophysin contains four transmembrane regions and a carboxyl-terminal domain having a novel repetitive structure. To elucidate the transmembrane organization of this protein in the synaptic vesicle, five antipeptide antibodies were raised. The site-specific antibodies were used to map the cognate sequences to the cytoplasmic or intravesicular side of the synaptic vesicle membrane by determining the susceptibility of the epitopes to proteolysis. The results confirm a topographic model for synaptophysin in which the protein spans the vesicle membrane four times, with both the amino and carboxyl terminus being cytoplasmic. In addition, the evolutionary conservation of the synaptophysin domains was addressed as a function of their membrane localization. To this end the primary structure of bovine synaptophysin was determined. Sequence comparisons between bovine, rat, and human synaptophysin revealed that only the intravesicular loops showed a significant number of amino acid substitutions (22%), while the transmembrane regions and cytoplasmic sequences were highly conserved (3% substitutions). These results depict synaptophysin as a protein with multiple membrane spanning regions whose functional site is likely to reside in highly conserved intramembranous and cytoplasmic sequences.  相似文献   

2.
Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.  相似文献   

3.
H Rehm  B Wiedenmann    H Betz 《The EMBO journal》1986,5(3):535-541
Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X-100 and purified by immunoaffinity or ion-exchange chromatography. From gel permeation and sucrose-density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross-linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ -o-phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri- and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N-glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium-binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+-dependent neurotransmitter release.  相似文献   

4.
Synaptophysin, an integral membrane protein of small synaptic vesicles, was expressed by transfection in fibroblastic CHO-K1 cells. The properties and localization of synaptophysin were compared between transfected CHO-K1 cells and native neuroendocrine PC12 cells. Both cell types similarly glycosylate synaptophysin and sort it into indistinguishable microvesicles. These become labeled by endocytic markers and are primarily concentrated below the plasmalemma and at the area of the Golgi complex and the centrosomes. A small pool of synaptophysin is transiently found on the plasma membrane. In CHO-K1 cells synaptophysin co-localizes with transferrin that has been internalized by receptor-mediated endocytosis. These findings suggest that synaptophysin in transfected CHO-K1 cells and neuroendocrine PC12 cells is directed into a pathway of recycling microvesicles which, in CHO cells, is shown to coincide with that of the transferrin receptor. They further indicate that fibroblasts have the ability to sort a synaptic vesicle membrane protein. Our results suggest a pathway for the evolution of small synaptic vesicles from a constitutively recycling organelle which is normally present in all cells.  相似文献   

5.
Serotonin organelles of rabbit platelets contain synaptophysin   总被引:1,自引:0,他引:1  
Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.  相似文献   

6.
Abstract: "Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS. a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation. we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine β-hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]-Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin: however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of ∼ 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine β-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.  相似文献   

7.
A D Linstedt  R B Kelly 《Neuron》1991,7(2):309-317
The targeting of synaptophysin, a major synaptic vesicle protein, in transfected nonneuronal cells has important implications for synaptic vesicle biogenesis, but has proved controversial. We have analyzed four transfected cell types by differential centrifugation and velocity gradient sedimentation to determine whether synaptophysin is targeted to endosomes or to synaptic vesicle-like structures. Synaptophysin was recovered only in vesicles that sedimented more rapidly than synaptic vesicles. The synaptophysin-containing vesicles were labeled if a surface-labeled cell was warmed to 37 degrees C, comigrated with transferrin receptor-containing vesicles on velocity and density gradients, and could be completely immunoadsorbed by anti-LDL receptor tail antibodies. These data demonstrate that synaptophysin was targeted to the early endocytotic pathway in the transfected cells and are inconsistent with the suggestion that synaptophysin expression induces a novel population of vesicles. Targeting of synaptophysin to early endosomes implicates their role in synaptic vesicle biogenesis.  相似文献   

8.
Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.  相似文献   

9.
Proteins conferring immunity against pore-forming colicins are localized in the Escherichia coli inner membrane. Their protective effects are mediated by direct interaction with the C-terminal domain of their cognate colicins. Cai, the immunity protein protecting E. coli against colicin A, contains four cysteine residues. We report cysteine cross-linking experiments showing that Cai forms homodimers. Cai contains four transmembrane segments (TMSs), and dimerization occurs via the third TMS. Furthermore, we observe the formation of intramolecular disulfide bonds that connect TMS2 with either TMS1 or TMS3. Co-expression of Cai with its target, the colicin A pore-forming domain (pfColA), in the inner membrane prevents the formation of intermolecular and intramolecular disulfide bonds, indicating that pfColA interacts with the dimer of Cai and modifies its conformation. Finally, we show that when Cai is locked by disulfide bonds, it is no longer able to protect cells against exogenous added colicin A.  相似文献   

10.
Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.  相似文献   

11.
Synaptophysin is one of the major integral membrane proteins of the small (30–50 nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. the behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9 nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.This research was supported by the DFG-SFB 317  相似文献   

12.
Subcellular fractionation of rabbit optic nerve resolves three populations of membranes that are rapidly labelled in the axon. The lightest membranes are greater than 200 nm and are relatively immobile. The intermediate density membranes consist of 84 nm vesicles which disappear from the nerve with kinetics identical to those of the rapid component. A third population of membranes, displaying a distinct protein profile, is present in the most dense region of the gradient. Immunological characterization of these membranes suggests the following. (1) The lightest peak contains rapidly transported glucose transporter and most of the total glucose transporters present in the nerve; this peak is therefore enriched in axolemma. (2) The intermediate peak contains rapidly transported glucose transporters and synaptophysin, an integral synaptic vesicle protein, and about half of the total synaptophysin; this peak therefore contains transport vesicles bound for both the axolemma and the nerve terminal, and these subpopulations can be separated by immunoadsorption with specific antibodies against the aforementioned proteins. (3) The heaviest peak contains rapidly transported synaptophysin and tachykinin neuromodulators and about half of the total synaptophysin, and 80% of the total tachykinins present in the nerve; this peak appears to represent a class of synaptic vesicle precursor bound for the nerve terminal exclusively. (4) Synaptophysin is present in the membranes of vesicles carrying tachykinins. (5) Both the intermediate and the heaviest peaks are enriched in kinesin heavy chain, suggesting that both vesicle classes may be transported by the same mechanism.  相似文献   

13.
Increased synaptophysin expression through whisker stimulation in rat   总被引:2,自引:0,他引:2  
1. Synaptophysin is responsible for the cycling of the synaptic vesicles containing the neurotransmitter, and it can be phosphorylated.2. This study examined whether repeated whisker stimulation alters the expression of synaptophysin mRNA in the rat barrel cortex, and found induced expression of synaptophysin mRNA in the contralateral barrel cortex compared to that in the ipsilateral hemisphere.3. This result suggests that synaptophysin is involved in the modulation of the synaptic plasticity.  相似文献   

14.
《The Journal of cell biology》1990,111(5):2041-2052
We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane.  相似文献   

15.
Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.  相似文献   

16.
The biogenesis of synaptic-like microvesicles (SLMVs) in neuroendocrine cells was investigated by studying the traffic of newly synthesized synaptophysin to SLMVs in PC12 cells. Synaptophysin was found to be sulfated, which facilitated the determination of its exit route from the trans-Golgi network (TGN). Virtually all [35S]sulfate-labeled synaptophysin was found to leave the TGN in vesicles which were indistinguishable from constitutive secretory vesicles but distinct from immature secretory granules and SLMVs. [35S]sulfate-labeled synaptophysin was rapidly transported from the TGN to the cell surface, with a t1/2 of approximately 10 min in resting cells. After arrival at the cell surface, [35S]sulfate-labeled synaptophysin cycled for at least 1 h between the plasma membrane and an intracellular compartment likely to be the early endosome. Up to approximately 40% of the [35S]sulfate-labeled synaptophysin eventually (after 3 h and later) reached SLMVs, which could be distinguished from the other post-TGN compartments by their lower buoyant density in a sucrose gradient and their selective inclusion upon permeation chromatography using a controlled-pore glass column. Our results suggest that newly synthesized membrane proteins of SLMVs in neuroendocrine cells, and possibly of small synaptic vesicles in neurons, reach these organelles via the TGN----plasma membrane----early endosome.  相似文献   

17.
The effects of ascorbic acid (AsA) on the formation process for a heat-induced gel offish meat (kamaboko) were examined. An investigation of the bonds influenced by adding AsA indicates that the aggregation of protein by noncovalent binding decreased and that by cross-linking, except for disulfide bonding, significantly increased in comparison with the control during a 30- min incubation at 40°C (suwari process). The results from the same investigation on a heat-induced gel incubated at 90°C for 30 min without using the suwari process, and the effects of AsA on the activity of transglutaminase indicate that this difference was derived not from activation of the enzyme by AsA but from the direct effect of AsA on the proteins. No effect of AsA on the increase in surface hydrophobicity of crude actomyosin at 40 and 90°C was found. Moreover, when the surimi with modified sulfhydryl groups was used, the disappearance of aggregation influenced by adding AsA and an accumulation of aggregates by noncovalent bonding during the formation of a heat-induced gel occured. These results suggest that polymerization during the formation of a heat-induced gel proceeded as follows: native proteins were first aggregated by noncovalent bonding, next by disulfide bonding, and finally by cross-linking apart from disulfide bonding, and that AsA improved the quality of a heat-induced gel by accelerating the formation of disulfide bonds.  相似文献   

18.
《The Journal of cell biology》1988,107(6):2717-2727
Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of alpha-latrotoxin in Ca2+-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, alpha-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of alpha-latrotoxin in the presence of 1.8 mM extracellular Ca2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.  相似文献   

19.
Domain structure of synaptotagmin (p65)   总被引:25,自引:0,他引:25  
Synaptotagmin (p65) is an abundant and evolutionarily conserved protein of synaptic vesicles that contains two copies of an internal repeat homologous to the regulatory region of protein kinase C. In the current study, we have investigated the biochemical properties of synaptotagmin, demonstrating that it contains five protein domains: an intravesicular amino-terminal domain that is glycosylated but lacks a cleavable signal sequence; a single transmembrane region; a sequence separating the transmembrane region from the two repeats homologous to protein kinase C; the two protein kinase C-homologous repeats; and a conserved carboxyl-terminal sequence following the two repeats homologous to protein kinase C. Sucrose density gradient centrifugations and gel electrophoresis indicate that synaptotagmin monomers associate into dimers and are part of a larger molecular weight complex. A sequence predicted to form an amphipathic alpha-helix that may cause the stable dimerization of synaptotagmin is found in its third domain between the transmembrane region and the protein kinase C-homologous repeats. Synaptotagmin contains a single hypersensitive proteolytic site that is located immediately amino-terminal to the amphipathic alpha-helix, suggesting that synaptotagmin contains a particularly exposed region as the peptide backbone emerges from the dimer. Finally, subcellular fractionation and antibody bead purification demonstrate that synaptotagmin co-purifies with synaptophysin and other synaptic vesicle markers in brain. However, in the adrenal medulla, synaptotagmin was found in both synaptophysin-containing microvesicles and in chromaffin granules that are devoid of synaptophysin, suggesting a shared role for synaptotagmin in the exocytosis of small synaptic vesicles and large dense core catecholaminergic vesicles.  相似文献   

20.
Synaptophysin is one of the most abundant membrane proteins of small synaptic vesicles. In mature nerve terminals it forms a complex with the vesicular membrane protein synaptobrevin, which appears to modulate synaptobrevin's interaction with the plasma membrane-associated proteins syntaxin and SNAP25 to form the SNARE complex as a prerequisite for membrane fusion. Here we show that synaptobrevin is preferentially cleaved by tetanus toxin while bound to synaptophysin or when existing as a homodimer. The synaptophysin/synaptobrevin complex is, however, not affected when neuronal secretion is blocked by botulinum A toxin which cleaves SNAP25. Excessive stimulation with alpha-latrotoxin or Ca(2+)-ionophores dissociates the synaptophysin/synaptobrevin complex and increases the interaction of the other SNARE proteins. The stimulation-induced dissociation of the synaptophysin/synaptobrevin complex is not inhibited by pre-incubating neurones with botulinum A toxin, but depends on extracellular calcium. However, the synaptophysin/synaptobrevin complex cannot be directly dissociated by calcium alone or in combination with magnesium. The dissociation of synaptobrevin from synaptophysin appears to precede its interaction with the other SNARE proteins and does not depend on the final fusion event. This finding further supports the modulatory role the synaptophysin/synaptobrevin complex may play in mature neurones.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号