Holt Matthew, Riedel Dietmar, Stein Alexander, Schuette Christina, Jahn Reinhard
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D37077 Göttingen, Germany.
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D37077 Göttingen, Germany.
Curr Biol. 2008 May 20;18(10):715-722. doi: 10.1016/j.cub.2008.04.069.
In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays.
By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin.
Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.
在神经元中，神经递质的释放通过突触小泡与质膜的融合来实现。已经鉴定出了这个过程所需的许多蛋白质，其中SNARE蛋白 syntaxin 1、SNAP - 25和突触囊泡蛋白被认为构成了核心融合机制。然而，我们对单个蛋白质 - 蛋白质相互作用的理解与完整突触制剂中扰动所揭示的这些蛋白质的功能之间仍存在很大差距。为了弥合这一差距，我们在融合实验中使用了纯化的突触小泡以及含有核心组成的SNARE蛋白作为反应伙伴的人工膜。
通过使用互补的实验方法，我们表明突触小泡与含有质膜蛋白 syntaxin 1和SNAP - 25的蛋白脂质体组成性地且高效地融合。融合受到梭菌神经毒素的抑制，并且涉及SNARE复合物的形成。尽管存在内源性突触结合蛋白，但即使脂质体膜中存在磷脂酰肌醇4,5 - 二磷酸，Ca(2+)也不会增强融合。相反，融合动力学主要由突触囊泡蛋白可利用的游离 syntaxin 1/SNAP - 25受体位点决定。
突触小泡是组成性激活的融合机器，仅需突触囊泡蛋白即可发挥活性。显然，融合的最后一步不涉及其他囊泡成分的调节活性，尽管这些成分可能参与调节早期过程。这对于胞吐作用的钙依赖性调节尤为重要，除了突触结合蛋白外，胞吐作用还需要囊泡膜中不存在的其他因素。本文描述的体外系统为阐明此类调节事件的分子细节提供了理想的起点。
