Functional mapping of SNARE-dependent trafficking and fusion

描述（由申请人提供）：本研究计划的总体目标是进一步了解脂肪细胞内细胞内运输、胰岛素刺激的葡萄糖转运蛋白（GLUT4）从细胞内储存位点到质膜的易位和融合的基本分子机制。尽管在GLUT4易位的胰岛素信号通路方面已经取得了重大进展，但关于特定细胞内运输步骤中涉及的调节事件和蛋白质的信息很少。此外，这种或任何其他系统的生物膜融合的基本生物物理机制尚未阐明。最近，我们发现新合成的GLUT4蛋白通过高尔基复合体运输，直接进入胰岛素反应性储存室，而无需首先通过质膜并进行内吞作用。基于GLUT4生物合成和细胞内分选的时间特性以及siRNA基因沉默的使用，我们设计了一种新的范式来研究GLUT4生命周期中不同膜运输步骤中起作用的靶向机制和囊泡运输事件。基于这些数据，我们建议使用siRNA结合GLUT4报告结构的时间表达，将参与生物合成分选的蛋白质从退出胰岛素反应室所需的蛋白质（2类）、内吞噬作用所需的蛋白质（3类）和再循环回胰岛素反应室所需的蛋白质（4类）功能定位到胰岛素反应室（1类）。我们建议使用这种方法来识别和分类生物膜融合所需的SNARE家族蛋白质的特定要求。与此同时，我们已经开始利用GLUT4运输细胞生物学方法结合磷脂- snare蛋白相互作用的生物物理分析来揭示snare依赖融合过程本身的生化性质。我们的初步数据表明，质膜Syntaxins （Stx）和VAMP的近膜结构域（JMD）都需要通过与酸性磷脂的静电相互作用来完成这一过程。为了解决融合孔形成的机制，我们将使用一系列生物物理学和细胞生物学方法来确定膜静电和串联VAMP2色氨酸在这一关键生物过程中的作用。

DESCRIPTION (provided by applicant): The overall objective of this research proposal is to further our understanding of the basic molecular mechanisms accounting for the intracellular trafficking, insulin-stimulated translocation and fusion of the insulin-responsive glucose transporter (GLUT4) from intracellular storage sites to the plasma membrane in adipocytes. Although significant progress has been made in terms of the insulin signaling pathways responsible for GLUT4 translocation, there is little information with regard to the regulatory events and proteins involved in specific intracellular trafficking steps. Moreover, the basic biophysical mechanism of biological membrane fusion for this, or any other system, has not been elucidated. Recently, we have found that newly synthesized GLUT4 protein traffics through the Golgi complex and directly enters the insulin-responsive storage compartment without first transiting the plasma membrane and undergoing endocytosis. Based upon the temporal properties of GLUT4 biosynthesis and intracellular sorting coupled with the use of siRNA gene silencing, we have devised a novel paradigm for examining the targeting machinery and vesicle trafficking events that function at different membrane transport steps in the GLUT4 lifecycle. Based upon these data we propose to use siRNA coupled with the temporal expression of GLUT4 reporter constructs to functionally map proteins involved in the biosynthetic sorting to the insulin-responsive compartment (Class 1) from those required for the exit from this compartment (Class 2), those required for endocytosis (Class 3) and those required for recycling back to the insulin-responsive compartment (Class 4). We propose to use this approach to identify and classify the specific requirements for the SNARE family of proteins required for biological membrane fusion. In parallel, we have begun to unravel the biochemical nature of the SNARE-dependent fusion process itself using a combination of GLUT4 trafficking cell biological approach coupled with biophysical analysis of phospholipid-SNARE protein interactions. Our preliminary data has demonstrated that the juxtamembrane domain (JMD) of both plasma membrane Syntaxins (Stx) and VAMP are required for this process through electrostatic interactions with acidic phospholipids. To address the mechanism of fusion pore formation, we will use a series of biophysical and cell biological approaches to determine the role of membrane electrostatics and the tandem VAMP2 tryptophans in this key biological process.