电子显微镜观察SNARE核心复合体的结构

作为神经突触中介导囊泡融合的核心蛋白,SNARE(soluble NSF attachment protein receptors)复合体由VAMP、syntaxin和SNAP25三种蛋白组成。而由其可溶性部分组成的SNARE核心复合体是发挥融合功能的关键部分。通过表达SNARE蛋白,应用分子筛和离子交换柱等纯化方法,重组得到了高纯度的SNARE核心复合体,并分析了其在水溶液中的稳定性。最后,采用负染电镜观察方法得到了该复合体的二维平均结构。在水溶液中,SNARE复合体核心部分为2.5 nm宽、12 nm长的棒状结构。     

植物SNARE蛋白的结构与功能

在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的,SNARE蛋白的结构高度保守.研究发现,植物中的SNARE蛋白促进植物细胞板形成,能与离子通道蛋白相互作用,有利于植物的正常生长发育,能提高植物的抗病性及参与植物的向重力性作用.应用基因组学和蛋白质组学技术结合细胞学水平上的分析方法有助于深入揭示植物SNARE蛋白家族成员的功能,明确SNARE蛋白在信号转导途径中的作用,阐明动植物免疫系统的区别和联系.     

植物SNARE 蛋白的结构与功能

在真核生物细胞囊泡运输过程中的膜融合主要是由SNARE蛋白介导的, SNARE蛋白的结构高度保守。研究发现, 植物中的SNARE蛋白促进植物细胞板形成, 能与离子通道蛋白相互作用, 有利于植物的正常生长发育, 能提高植物的抗病性及参与植物的向重力性作用。应用基因组学和蛋白质组学技术结合细胞学水平上的分析方法有助于深入揭示植物SNARE蛋白家族成员的功能, 明确SNARE蛋白在信号转导途径中的作用, 阐明动植物免疫系统的区别和联系。     

Gβγ SNARE Interactions and Their Behavioral Effects

Presynaptic terminals possess interlocking molecular mechanisms that control exocytosis. An example of such complexity is the modulation of release by presynaptic G Protein Coupled Receptors (GPCRs). GPCR ubiquity at synapses—GPCRs are present at every studied presynaptic terminal—underlies their critical importance in synaptic function. GPCRs mediate presynaptic modulation by mechanisms including via classical Gα effectors, but membrane-delimited actions of Gβγ can also alter probability of release by altering presynaptic ionic conductances. This directly or indirectly modifies action potential-evoked presynaptic Ca²⁺ entry. In addition, Gβγ can interact directly with SNARE complexes responsible for synaptic vesicle fusion to reduce peak cleft neurotransmitter concentrations during evoked release. The interaction of Gβγ with SNARE is displaced via competitive interaction with C2AB-domain containing calcium sensors such as synaptotagmin I in a Ca²⁺-sensitive manner, restoring exocytosis. Synaptic modulation of this form allows selective inhibition of postsynaptic receptor-mediated responses, and this, in combination with Ca²⁺ sensitivity of Gβγ effects on SNARE complexes allows for specific behavioral outcomes. One such outcome mediated by 5-HT receptors in the spinal cord seen in all vertebrates shows remarkable synergy between presynaptic effects of Gβγ and postsynaptic 5-HT-mediated changes in activation of Ca²⁺-dependent K⁺ channels. While acting through entirely separate cellular compartments and signal transduction pathways, these effects converge on the same effect on locomotion and other critical functions of the central nervous system.

     

SNARE蛋白调控细胞自噬的分子机制

细胞自噬是细胞在面对内外部环境压力的情况下, 为了自身的稳定而采取的一种降解内部及外来入侵物质的机制。SNARE(Soluble N-ethylmaleimide-sensitive factor attachment protein receptors)假说指出SNARE蛋白在细胞物质运输以及特异性膜融合过程中具有重要作用, 揭示了细胞正常生理活动有序进行的分子机制。由于细胞自噬涉及从自噬体的形成到自噬体溶酶体的融合等诸多膜融合的过程, 因此, 文章对近年来SNARE蛋白在调控细胞自噬过程的研究进展进行了综述。     

Are neuronal SNARE proteins Ca2+ sensors?

The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.     

拟南芥SNARE因子在膜泡运输中的功能

高等植物细胞含有复杂的内膜系统,通过其特有的膜泡运输机制来完成细胞内和细胞间的物质交流。膜泡运输主要包括运输囊泡的出芽、定向移动、拴留和膜融合4个过程。这4个过程受到许多因子的调控,如Coat、SM、Tether、SNARE和Rab蛋白等,其中SNARE因子在膜融合过程中发挥重要功能。SNARE因子是小分子跨膜蛋白,分为定位于运输囊泡上的v-SNARE和定位于靶位膜上的t-SNARE,两类SNARE结合形成SNARE复合体,促进膜融合的发生。SNARE蛋白在调控植物体生长发育以及对外界环境响应等生理过程中起重要作用。该文对模式植物拟南芥（Arabidopsis thaliana）SNARE因子的最新细胞内定位和功能分析等研究进展进行了概述。     

SNARE Proteins-Why So Many,Why So Few?

Abstract: Both trafficking and secretion critically depend on accurate and specific membrane recognition and fusion. A key step in these processes is the assembly of a complex consisting of a small number of proteins, i.e., the exocytic core complex. In nerve terminals, this set consists of VAMP and synaptotagmin, which reside at membranes of synaptic vesicles, and syntaxin and SNAP-25 at the plasma membrane. In this survey, different secretory systems that depend on the exocytic core proteins are considered. The possibility that specificity in membrane recognition and fusion is achieved by the numerous variants of proteins of the exocytic core is discussed. Variability of the core complex proteins is determined by the complexity of gene families, isoform-specific localization, and posttranslational modifications. Basic biochemical properties depend on specific isoforms, and the possible protein-protein interactions are determined, in turn, by the compatibility of different isoforms. A correlation between specific variants and distinct biochemical or cellular properties is shown. The outcome of this survey is that heterogeneity in secretion may be dictated by the large number of possible combinations of variants of only a few proteins.     

拟南芥SNARE因子在膜泡运输中的功能

高等植物细胞含有复杂的内膜系统, 通过其特有的膜泡运输机制来完成细胞内和细胞间的物质交流。膜泡运输主要包括运输囊泡的出芽、定向移动、拴留和膜融合4个过程。这4个过程受到许多因子的调控, 如Coat、SM、Tether、SNARE和Rab蛋白等, 其中SNARE因子在膜融合过程中发挥重要功能。SNARE因子是小分子跨膜蛋白, 分为定位于运输囊泡上的v-SNARE和定位于靶位膜上的t-SNARE, 两类SNARE结合形成SNARE复合体, 促进膜融合的发生。SNARE蛋白在调控植物体生长发育以及对外界环境响应等生理过程中起重要作用。该文对模式植物拟南芥(Arabidopsis thaliana)SNARE因子的最新细胞内定位和功能分析等研究进展进行了概述。     

SNARE蛋白及其相关蛋白在精子顶体反应中的作用

精子顶体反应是指精子顶体外膜与精子质膜发生细胞内多点融合的反应,其过程属于特殊的胞吐过程。精子顶体反应是一个复杂严谨的生理过程,不仅需要诱导剂的诱导,还需要多种相关膜融合蛋白的参与。SNARE蛋白及其相关蛋白能够调控哺乳动物细胞内的融合,尤其在精子顶体反应过程中发挥着重要作用。其中SNARE蛋白是核心成分,与其相关蛋白相互作用,共同参与精子顶体反应过程。现主要对SNAREs、Rab、Munc-18、complexin、synaptotagmin和α-SNAP等蛋白质在精子顶体反应中的作用进行概述。     

SNARE complexes and neuroexocytosis: how many, how close?

Regulated secretion is an essential process in all eukaryotic cells. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of a SNARE complex formed by the coil-coiling of three proteins: SNAP-25, syntaxin and VAMP/synaptobrevin. It seems that SNARE complexes assemble together in rosette-shaped super-complexes but there is controversy on the actual number (N) of copies of SNARE complexes that are necessary to mediate exocytosis. We discuss attempts to determine the value of N and suggest that N varies with the type of exocytic vesicles. In addition, we propose that the N value in neuroexocytosis can be estimated by the comparative use of different types of botulinum neurotoxins.     

酿酒酵母全基因组SNARE蛋白的亚细胞定位研究

在真核细胞中,内质网、高尔基体、质膜等膜结构间的蛋白质运输主要通过囊泡出芽和融合实现。SNARE蛋白家族在介导囊泡与目的膜结构融合过程中发挥关键作用。在模式生物酿酒酵母中,对全基因组SNARE蛋白的系统研究仍有不足。此研究构建了一套用于标记酿酒酵母基因组全部24种SNARE的工具质粒。该系列质粒既能呈现出良好的定位特征,又避免了过度表达造成的定位异常。通过与细胞器标记共定位验证了SNARE蛋白的亚细胞定位。结果发现3种SNARE的定位与之前报道不符:Bos1定位于早高尔基体,Snc1和Bet1定位于晚高尔基体/早内体。另外,Sec9定位于芽尖和芽颈,这是首次观察到Sec9在活酵母细胞中的定位。这项工作首次全面的检验了酵母SNARE家族蛋白的亚细胞定位,为后续SNARE蛋白功能研究提供了新线索,并为相关研究提供了一套工具质粒。     

What is the role of SNARE proteins in membrane fusion?

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion "machine." Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.     

Is lipid flippase activity of SNARE transmembrane domains required for membrane fusion?

It has been suggested that lipids translocate between the outer and inner leaflets of fusing membranes, or flip-flop, to facilitate changes in bilayer leaflet areas at various stages of fusion. Here, we investigated the lipid flip activity of synthetic peptides that mimic SNARE transmembrane domains (TMDs). These peptides indeed induce flip of marker lipids. However, mutations that reduce flip activity do not diminish fusogenicity and cholesterol blocks flip much more efficiently than fusion. Therefore, our data do not support a role for flip in membrane fusion. On the other hand, the ability of SNARE TMDs to catalyze flip is consistent with a role of SNAREs in biogenic lipid flip.     

烤烟SNARE蛋白基因T-NTGP2全长cDNA克隆与生物信息学分析

从烤烟品种南江3号(Nicotiana tobacum cv.Nanjiang-3)均一化cDNA文库中克隆获得一个SNARE蛋白同源性较高的cDNA全长序列,命名为T-NTGP2,GenBank中的登录号为HO663897.利用ORF finder和ProtParam软件分析显示,它包含一个长度为597 bp的完整开放读码框,编码一个含有199个氨基酸、等电点为6.96、分子量为22.59 kD的蛋白.Blast搜索结果及进化分析结果表明,烤烟T-NTGP2基因编码的蛋白与烤烟、杨树和蓖麻的Ykt6蛋白具有较高的同源性,其一致性分别为99％、91％和91％.该基因编码的蛋白具有一个典型的VAMP基元,同源分析表明该蛋白属于SNAREs中特殊longins中的Ykt6蛋白.     

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.     

A legend of the SNARE complex and synaptotagmin—the insight into synaptic transmission

The 2013 Nobel Prize in Physiology or Medicine honors three scientists,James Rothman,Randy Schekman and Thomas Südhof,whose work revealed the mystery of the vesicle trafficking,i.e.,how cells,from yeast to mammals,organize their transport systems.In a neuroscience perspective,this decades-long story might be described as a legend     

Is Assembly of the SNARE Complex Enough to Fuel Membrane Fusion?

The three key players in the exocytotic release of neurotransmitters from synaptic vesicles are the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin 2, syntaxin 1a, and SNAP-25. Their assembly into a tight four-helix bundle complex is thought to pull the two membranes into close proximity. It is debated, however, whether the energy generated suffices for membrane fusion. Here, we have determined the thermodynamic properties of the individual SNARE assembly steps by isothermal titration calorimetry. We found extremely large favorable enthalpy changes counterbalanced by positive entropy changes, reflecting the major conformational changes upon assembly. To circumvent the fact that ternary complex formation is essentially irreversible, we used a stabilized syntaxin-SNAP-25 heterodimer to study synaptobrevin binding. This strategy revealed that the N-terminal synaptobrevin coil binds reversibly with nanomolar affinity. This suggests that individual, membrane-bridging SNARE complexes can provide much less pulling force than previously claimed.The molecular machinery that drives the Ca²⁺-dependent release of neurotransmitters from synaptic vesicles is studied intensively. Three key players in the underlying exocytotic fusion of the vesicle with the plasma membrane are the proteins synaptobrevin 2/VAMP2 (vesicle-associated membrane protein), syntaxin 1a, and SNAP-25³ (for review, see Refs. 1-7). They belong to the so-called SNARE protein family, the members of which are involved in all vesicle fusion steps in the endocytic and secretory pathway. In general, SNARE proteins are relatively small, tail-anchored membrane proteins. Their key characteristic is the so-called SNARE motif, an extended stretch of heptad repeats that is usually connected to a single transmembrane domain by a short linker. Syntaxin and synaptobrevin each contain a single SNARE motif, whereas SNAP-25 contains two SNARE motifs connected by a palmitoylated linker region serving as a membrane anchor. The SNARE motifs of the three proteins assemble into a very tight four-helix bundle between opposing membranes; during this process the plasma membrane proteins syntaxin and SNAP-25 provide the binding site for the vesicular synaptobrevin. Formation of this complex is accompanied by extensive structural rearrangements (8-10). Based on these findings, it was put forward that the formation of the SNARE bundle provides the energy that drives membrane fusion. As the bundle is oriented in parallel, it is thought that formation of this complex starts from the membrane-proximal N termini and proceeds toward the C-terminal membrane anchors, effectively pulling the membranes together (the “zipper” model) (11). Although the zipper scenario is intuitive, it has been difficult to demonstrate directly.A decade ago it was shown that the three neuronal SNARE proteins are sufficient to fuse artificial vesicles (12). However, this reductionist approach yields rather slow fusion rates (12-14). Over the years various different end products of SNARE catalysis (complete fusion, hemifusion, and only tethered membranes) have been reported (15-19). These unsatisfactory results have fueled the debate over whether the assembly process indeed provides enough impetus to fuse bilayers. Not surprisingly, an alternative scenario has been put forward in which repulsive forces between membranes bring the SNARE assembly to a grinding halt. According to this idea, other factors like the Ca²⁺ sensor synaptotagmin or the small soluble protein complexin are needed to induce membrane merging (20-22).In simple terms, to find out whether the SNARE complex assembly is enough for membrane fusion, only the amount of energy released during complex formation and the amount of energy needed for membrane fusion need to be compared. However, the physics of membrane fusion are very complicated, and it is even more challenging to understand how proteins modulate the process. The free energy for bilayer fusion in an aqueous environment is not very high, but fusion is thought to require a large activation energy of about 40 k_BT, as two charged membranes have to be brought into close apposition. According to a theoretical model, the apposing membranes then need to be modified into a stalk-like configuration. Before fusion occurs, the process is thought to pass through a hemifusion intermediate in which only the outer monolayers are merged (for review, see Refs. 23 and 24). The role of fusion proteins is thought to lower the energy barrier for membrane fusion, but understanding how they modulate the lipid membrane and how their conformational changes are translated into a mechanical force is still in its infancy. It is not clear, for instance, whether SNARE-catalyzed fusion indeed proceeds through a stalk-like structure or just locally alters the membranes, a mechanism that might need much less activation energy.As the folding and unfolding transitions of the ternary SNARE complex exhibit a marked hysteresis (25), the question of how much energy is released during complex formation has been difficult to answer as well. To avoid the quasi-irreversibility of the process, the problem has been elegantly tackled by atomic force microscopy by two different research groups (26-28). In these experiments individual complexes affixed to solid supports were ruptured, yielding energy values of 43 and 33 k_BT. In another approach, which used a surface-force apparatus (SFA), a comparable energy of 35 k_BT has recently been determined (29). Strikingly, these values appear to correspond closely with the activation energy needed to fuse two membranes, substantiating the view that SNAREs are nano-fusion machineries. However, one should be cautious about the conclusion that these sophisticated procedures in fact yield the genuine SNARE assembly energy. For example, with the SFA approach, the number of complexes had to be deduced rather indirectly to estimate the free energy. Moreover, these approaches offered only indirect information about the reaction pathway.In this study we set out to determine the SNARE assembly energy more directly by using isothermal titration calorimetry (ITC) complemented by kinetic measurements. ITC is a powerful technique for studying the thermodynamics of macromolecular interactions by directly measuring the heat changes associated with complex formation, which at constant pressure is equal to the enthalpy change (ΔH). The titration approach also yields the stoichiometry (n), the entropy change (ΔS), and the association constant (K_A) of the reaction. We studied the consecutive reaction steps individually to gain deeper insights into the rugged energy landscape of complex formation. To study synaptobrevin binding in isolation, we used a stabilized syntaxin-SNAP-25 heterodimer, which has been shown to greatly accelerate liposome fusion rates (30). This strategy revealed that the N-terminal coil of synaptobrevin binds reversibly, making it feasible to access the free energy of SNARE assembly. Overall, our results suggest that individual SNARE complexes might provide much less pulling energy than previously claimed.     

The Arabidopsis R‐SNARE protein YKT61 is essential for gametophyte development

Gametophyte development is a pre‐requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N‐ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss‐of‐function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three‐base‐pair deletion in YKT61 in the ykt61‐3 mutant resulted in a single‐amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N‐terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9‐mediated editing and expression from a Cas9‐resistant transgene.