NSF-mediated disssembly of SNARE complexes

精确的膜运输对于细胞功能至关重要，从细胞内蛋白质运输到靶向分泌和神经递质释放。本研究计划的主要目的是了解如何实现这一规定。具体来说，我们正在研究膜融合机制的关键组成部分，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，因为它控制着神经递质的释放。膜融合是由囊泡和靶膜上SNARE蛋白之间形成的线圈状复合物驱动的，NSF被认为调节它们的组装和拆卸。NSF如何实现这些功能尚不清楚，尽管最近的研究表明NSF通过解开陷阱来解开它们。******在本基金的前期，我们发现哺乳动物NSF在神经元的几个位点受到酪氨酸激酶Fer和酪氨酸磷酸酶MEG2的酪氨酸磷酸化的影响。我们绘制了相关的酪氨酸残基，并测量了它们对分泌的贡献。这为美国国家科学基金会的监管提供了生物学证据，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/或解绕活性受NSF的酪氨酸磷酸化调节。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。我们将在酪氨酸磷酸化位点检测NSF中拟磷突变的snap结合和snare解离活性，以确定它们对NSF的调节模式。确定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fret的单分子检测方法来观察SNARE解离过程中NSF的活性。野生型和突变型NSF也将用于确定磷酸化对调节解扭活性的贡献。* * * 3。定义在SNARE分离过程中NSF和SNAREs的结构。我们将通过使用SNARE蛋白和另一种卷曲线圈蛋白GCN4之间的遗传融合来开发不可扭转的SNARE复合物。这将为NSF创造一个自杀底物，它将允许开始进行性unwind，但会将NSF困在复合体上。利用结构研究，我们将可视化NSF如何展开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其功能的外部控制。由于NSF在几乎所有生物体的细胞膜内交通、分泌、细胞间通讯和发育等过程中都是必不可少的，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. *****