Stoichiomeric biology and life cycle of the SNAREs

胞外SNAREs聚集在膜上，这一概念在迄今为止研究的所有膜蛋白中都是常见的。这对生物科学构成了新的挑战：首先，解开自由蛋白质与簇状蛋白质的作用和动力学，其次，理解流入簇状系统和流出簇状系统的流动。在这里，我们建议结合我们实验室的专业知识来研究SNARE的生命周期，不同SNARE种群之间的相对比例以及突触中SNARE蛋白质聚类/化学计量的基础知识。我们将确定突触中SNAREs的数量，它们的聚集，以及聚集蛋白和自由蛋白之间的交换，以及SNAREs进出突触的贩运。我们将确定突触功能特别需要的SNAREs的比例（活性池），我们将评估这些簇是否直接参与胞吐的功能单位。最后，我们将测试这些簇是否代表了分子的储备池，或者它们是否具有信号功能，例如集中辅助蛋白。我们期望揭示SNAREs的化学计量生物学和生命周期将大大促进我们对突触功能的理解。更重要的是，我们将首次了解生物膜中一组蛋白质的功能组织。

The exocytotic SNAREs are clustered in membranes, a concept which is common for essentially all membrane proteins investigated to date. This constitutes new challenges for biological sciences: first, to unravel the roles and dynamics of free versus clustered proteins and second, to understand both the flow into the cluster system and the flow out of the cluster system. Here, we propose to combine the expertise of our laboratories to study the life-cycle of SNAREs, the relative proportions between different SNARE-populations and basics of SNARE protein clustering/stoichiometry in the synapse. We will determine the numbers of SNAREs in the synapse, their clustering, as well as the exchange between clustered and free proteins, as well as the trafficking of SNAREs into and out of the synapse. We will determine the fraction of SNAREs specifically required for synapse function (the active pool), and we will assess whether the clusters are functional units directly involved in exocytosis. Finally, we will test whether the clusters represent reserve pools of molecules, or whether they have a signaling function, for example in concentrating accessory proteins. We expect that unraveling the stoichiometric biology and the life cycle of SNAREs will greatly advance our understanding of synaptic function. More importantly, we will obtain for the first time an understanding of the functional organization of a set of proteins in biological membranes.