NSF-mediated disssembly of SNARE complexes

精确的膜运输对细胞功能至关重要，从细胞内蛋白运输到靶向分泌和神经递质释放。这项研究计划的主要目的是了解这一规定是如何实现的。具体地说，我们正在研究膜融合机制的关键组件--ATPase N-乙基马来酰亚胺敏感因子(NSF)的功能。这种调节在大脑中极其重要，因为它控制着神经递质的释放。膜融合是由囊泡和靶膜上的SNARE蛋白之间形成卷曲的卷曲复合体来驱动的，NSF被认为调节它们的组装和拆解。NSF如何执行这些功能尚不清楚，尽管最近的研究表明，NSF通过解开陷阱来解开陷阱。*在这项资助的前一期中，我们发现哺乳动物的NSF在神经元中的几个位置受到酪氨酸激酶Fer和酪氨酸磷酸酶MEG2的酪氨酸磷酸化的影响。我们已经绘制了相关的酪氨酸残基图，并测量了它们对分泌的贡献。这为调控NSF提供了生物学证据，并为本拨款申请中提出的实验提供了基础。*我们假设NSF的SNARE结合和/或解离活性是由NSF的酪氨酸磷酸化调节的。我们建议通过完成以下目标来测试这些可能性：*1.确定NSF的SNAP结合或SNARE解离是否被磷酸化修饰。我们将检测NSF中拟磷酸突变在酪氨酸磷酸化位点的SNAP结合和SNARE解离活性，以确定它们对NSF的调节方式。*2.确定ATPase活性和NSF解旋酶活性的关系。我们将确定ATPase活性是协同发生的还是随机发生的，并开发一种基于单分子FRET的分析方法来可视化SNARE解离过程中的NSF活性。野生型和突变型NSF也将被用来确定磷酸化对调节解旋活性的贡献。*3.定义NSF的结构和陷阱解离过程中的陷阱。我们将利用SNARE蛋白和另一种卷曲蛋白Gcn4之间的基因融合来开发一种不扭曲的SNARE复合体。这将为NSF创造一种自杀式底物，允许开始进行解离，但会将NSF困在复合体上。利用结构研究，我们将可视化NSF如何解开SNARE复合体。了解NSF如何介导膜融合并识别调节它的蛋白质可以促进对其功能的外部控制。由于NSF在几乎所有生物的细胞膜运输、分泌、细胞间通讯和发育等过程中都是必不可少的，因此理解和控制这个重要的蛋白质对几乎所有生物学领域都具有重要意义。*****

Precise membrane traffic is essential for cellular functions ranging from intracellular protein transport to targeted secretion and neurotransmitter release. The major aim of this research program is to understand how this regulation is achieved. Specifically, we are investigating the function of a pivotal component of the membrane fusion machinery, the ATPase N-ethylmaleimide sensitive factor (NSF). This regulation is extremely important in the brain as it controls neurotransmitter release. Membrane fusion is driven by the formation of coiled-coil complexes between SNARE proteins on vesicles and target membranes, and NSF is thought to regulate their assembly and disassembly. How NSF carries out these functions remains unclear although recent studies have suggested that NSF unwinds the SNAREs by untwisting them. ******In the previous term of this grant we found that mammalian NSF is subject to tyrosine phoshphorylation at several sites in neurons by the tyrosine kinase Fer and the tyrosine phosphatase MEG2. We have mapped the tyrosine residues involved and measured their contribution to secretion. This provided the biological evidence for a regulation of NSF and provides the basis for experiments proposed within this grant application.******We hypothesize that SNARE binding and/or unwinding activity of NSF is regulated by tyrosine phosphorylation of NSF. We propose to test these possibilities by completing the following aims:***1. Determine if aSNAP binding or SNARE dissociation by of NSF is modified by phosphorylation. We will examine SNAP-binding and SNARE-dissociation activities of phosphomimetic mutations in NSF at sites of tyrosine phosphorylation to determine their modes of NSF regulation.***2. Determine the relationship between ATPase activity and NSF helicase activity. We will determine if ATPase activity occurs cooperatively or randomly and develop a single molecule FRET-based assay to visualize NSF activity during SNARE dissociation. Wild type and mutant NSF will also be used to determine the contribution of phosphorylation to regulating untwisting activity. ***3. Define the structure of NSF and SNAREs during SNARE dissociation. We will develop an untwistable SNARE complex by using genetic fusions between SNARE proteins and another coiled coil protein GCN4. This will create a suicide substrate for NSF that will allow processive unwinding to begin, but will trap the NSF on the complex. Using structural studies we will visualize how NSF unwinds SNARE complexes.******Understanding how NSF mediates membrane fusion and identifying proteins that regulate it could facilitate extrinsic control of its function. Since NSF is essential for processes ranging from intracellular membrane traffic, secretion, intercellular communication and development in virtually all living organisms, understanding and controlling this important protein will be significant to virtually all fields of biology. *****