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。

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.