NSF-mediated disssembly of SNARE complexes

精确的膜运输对于从细胞内蛋白质运输到靶向分泌和神经递质释放的细胞功能至关重要。 这项研究计划的主要目的是了解这种调节是如何实现的。 具体来说，我们正在研究膜融合机制的一个关键组成部分，ATP酶N-乙基马来酰亚胺敏感因子（NSF）的功能。 这种调节在大脑中非常重要，因为它控制神经递质的释放。 膜融合是由囊泡上的SNARE蛋白和靶膜之间形成卷曲螺旋复合物驱动的，NSF被认为调节它们的组装和分解。 NSF如何执行这些功能仍然不清楚，尽管最近的研究表明NSF通过解开它们来解开SNARE。** 在本授权的上一个学期，我们发现哺乳动物NSF在神经元中的几个位点受到酪氨酸激酶Fer和酪氨酸磷酸酶MEG 2的酪氨酸磷酸化。 我们绘制了酪氨酸残基参与和测量其分泌的贡献。这为NSF的一项法规提供了生物学证据，并为本资助申请中提出的实验提供了基础。我们假设SNARE结合和/或解旋活性的NSF调节的NSF的酪氨酸磷酸化。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aSNAP结合或SNARE解离是否被磷酸化修饰。 我们将检查NSF中酪氨酸磷酸化位点的磷酸化模拟突变的SNAP结合和SNARE解离活性，以确定它们的NSF调节模式。2.测定ATP酶活性与NSF解旋酶活性之间的关系。 我们将确定ATP酶活性是协同发生还是随机发生，并开发一种基于单分子FRET的测定方法来观察SNARE解离过程中的NSF活性。野生型和突变型NSF也将用于确定磷酸化对调节解旋活性的贡献。 *3。定义SNARE解离过程中NSF和SNARE的结构。 我们将通过使用SNARE蛋白和另一种卷曲螺旋蛋白GCN 4之间的基因融合来开发一种不可扭转的SNARE复合物。 这将为NSF创建一个自杀底物，其将允许进行性解旋开始，但将使NSF陷入复合物中。 使用结构研究，我们将可视化NSF如何解开SNARE复合物。了解NSF如何介导膜融合并识别调节它的蛋白质可以促进其功能的外部控制。 由于NSF对于几乎所有生物体的细胞内膜运输，分泌，细胞间通讯和发育过程都是必不可少的，因此了解和控制这种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. *****