Protein-Membrane Interactions in Regulated Exocytosis

描述(申请人提供)：调节性胞吐是一种依赖于刺激的膜融合事件，对一系列生理过程具有基本重要性。膜融合反应涉及大量的脂类重排，并受到强大的疏水作用力的反对。要发生核聚变，必须克服高能障碍。这项研究的总体目标是以葡萄糖转运蛋白GLUT4的转运为模型系统，确定膜双层重塑在克服胞外囊泡融合能量障碍中的功能作用。为了实现这一目标，我们将采用独特的互补方法组合，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被SNARE-SM复合体紧密对接的，为后续的膜重塑和合并步骤做准备。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如何通过质膜的局部重塑来调节。我们假设胞外囊泡与质膜的融合涉及一种新的双曲率诱导机制：当v-SNARE的两亲性基序弯曲囊泡膜时，C2结构域分子的疏水环穿透到质膜并诱导局部膜曲率。总之，这些薄膜弯曲活动有望在融合部位产生曲率应力，以克服融合反应的能量障碍。这项拟议的研究的成功完成将为胞外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膜融合的一般原理的范例。最终，我们的发现将有助于开发新的治疗策略，用于治疗与功能失调的胞吐相关的疾病，包括糖尿病、癫痫和免疫紊乱。

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.