Functional mapping of SNARE-dependent trafficking and fusion

描述（由申请方提供）：本研究提案的总体目标是进一步了解脂肪细胞中胰岛素应答性葡萄糖转运蛋白（GLUT 4）从细胞内储存位点到质膜的细胞内运输、胰岛素刺激易位和融合的基本分子机制。虽然在负责GLUT 4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控事件和蛋白质的信息很少。此外，生物膜融合的基本生物物理机制，这个系统，或任何其他系统，还没有得到阐明。最近，我们发现，新合成的GLUT 4蛋白通过高尔基复合体运输，并直接进入胰岛素响应的存储区室，而不首先通过质膜和进行内吞作用。基于GLUT 4生物合成和细胞内分选的时间特性，再加上使用siRNA基因沉默，我们设计了一种新的范式，用于检查在GLUT 4生命周期中不同膜转运步骤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与GLUT 4报告构建体的时间表达偶联，以将参与生物合成分选的蛋白质从退出胰岛素反应区室所需的蛋白质（第2类）、内吞作用所需的蛋白质（第3类）和再循环回胰岛素反应区室所需的蛋白质（第4类）功能性地映射到胰岛素反应区室（第1类）。我们建议使用这种方法来识别和分类的SNARE家族的生物膜融合所需的蛋白质的具体要求。与此同时，我们已经开始使用GLUT 4运输细胞生物学方法与磷脂-SNARE蛋白相互作用的生物物理分析相结合来解开SNARE依赖性融合过程本身的生物化学性质。我们的初步数据表明，质膜突触融合蛋白（Stx）和VAMP的质膜结构域（JMD）是通过与酸性磷脂的静电相互作用来实现这一过程所必需的。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VAMP 2双链体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.