Protein-Membrane Interactions in Regulated Exocytosis

描述（由申请人提供）：调节性胞吐是一种对一系列生理过程具有根本重要性的刺激依赖性膜融合事件。膜融合反应涉及大量的脂质重排，并受到强大的疏水力的反对。要使核聚变发生，必须克服一个高能势垒。这项研究的总体目标是确定膜双层重塑在克服胞吐囊泡融合的能量屏障中的功能作用，使用葡萄糖转运蛋白GLUT 4作为模型系统的贩运。为了实现这一目标，我们将采用一种独特的互补方法，包括生化重建，电子显微镜成像和生物物理测量。首先，我们将定义两个膜双层是如何通过SNARE-SM复合物紧密贴壁的，为膜重塑和合并的后续步骤做准备。接下来，我们将评估囊泡膜弯曲在融合反应中的功能作用。最后，我们将研究融合反应是如何通过质膜的局部重塑来调节的。我们推测，融合的胞吐囊泡与质膜涉及一种新的双曲率诱导机制：而两亲性基序的v-SNARE弯曲囊泡膜，疏水环的C2结构域分子渗透到质膜，并诱导局部膜曲率。总之，这些膜弯曲活动预计将在融合部位产生曲率应力，以克服融合反应的能垒。这项研究的成功完成将为胞吐囊泡融合的分子机制提供关键的见解。这项工作也将作为理解细胞内膜融合的一般原理的范例。最终，我们的研究结果将促进与功能失调的调节胞吐相关的疾病，包括糖尿病，癫痫和免疫疾病的新的治疗策略的发展。

DESCRIPTION (provided by applicant): Regulated exocytosis is a stimulus-dependent membrane fusion event of fundamental importance to a range of physiological processes. The membrane fusion reaction involves substantial lipid rearrangements and is opposed by the powerful hydrophobic force. For fusion to occur, a high energy barrier must be overcome. The overall goal of this proposed research is to determine the functional roles of membrane bilayer remodeling in overcoming the energy barrier of exocytic vesicle fusion, using the trafficking of the glucose transporter GLUT4 as a model system. To achieve this goal, we will employ a unique combination of complementary approaches including biochemical reconstitution, electron microscopic imaging, and biophysical measurements. First, we will define how two membrane bilayers are brought into close apposition by the SNARE-SM complex to prepare for the subsequent steps of membrane remodeling and merging. Next, we will assess the functional role of vesicle membrane bending in the fusion reaction. Finally, we will examine how the fusion reaction is regulated by local remodeling of the plasma membrane. We hypothesize that the fusion of exocytic vesicles with the plasma membrane involves a novel dual-curvature-induction mechanism: while the amphipathic motif of the v-SNARE bends the vesicle membrane, the hydrophobic loops of C2-domain molecules penetrate into the plasma membrane and induce local membrane curvature. Together, these membrane-bending activities are expected to create curvature stresses at the fusion sites to overcome the energy barrier for the fusion reaction. Successful completion of this proposed research will provide key insights into the molecular mechanisms of exocytic vesicle fusion. This work will also serve as a paradigm for understanding the general principles of intracellular membrane fusion. Ultimately, our findings will facilitate the development of novel therapeutic strategies for diseases associated with dysfunctional regulated exocytosis including diabetes, epilepsy, and immune disorders.