DYNAMIN'S FUNCTION IN ENDOCYTOSIS

DYNAMIN 的内吞作用

• 批准号: 6525736
• 负责人: Sean D Conner
• 金额: $ 4.62万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-10 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6525736
• 关键词: affinity chromatography; cell membrane; clathrin; complementary DNA; dynamin; endocytosis; guanosinetriphosphatases; immunofluorescence technique; laboratory rat; membrane reconstitution /synthesis; molecular cloning; mutant; protein binding; protein sequence; protein structure function; recombinant proteins; western blottings

The clathrin-mediated endocytic pathway is vital to a variety of biological processes ranging from synaptic vesicle recycling to nutrient uptake. In addition to the major coat protein complexes, the GTPase dynamin has an essential role in clathrin-coated vesicle (CCV) formation. Dynamin is currently postulated to function as a molecular `pinchase' that facilitates the budding of CCVs from the plasma membrane, however recent evidence from the Schmid lab supports its role as a key regulator of endocytosis. Thus our goals are to establish dynamin's role as a regulator of endocytosis by: l) defining molecules that are required for dynamin recruitment to clathrin-coated pits and 2) identifying upstream and downstream effectors of dynamin function. To accomplish these goals, we will take a strict biochemical approach using functionally relevant constructs to screen cellular extracts by affinity chromatography. Factors found to specifically interact with dynamin will be immediately tested for their ability to support discrete steps in vesicle formation using cell-free assays for endocytosis that faithfully reconstitute the production of CCVs in vitro. The clathrin-mediated endocytic pathway is fundamental and highly conserved, and since abnormal expression or mutations of some of the endocytic machinery proteins have been reported in human disease and development, information obtained from this project will not only add to our understanding of the basic mechanisms of clathrin-mediated endocytosis, but may provide insight into the development of certain disease types.

网格蛋白介导的内吞途径对于从突触囊泡回收到养分吸收的各种生物学过程至关重要。除了主要的外套蛋白复合物外，GTPase Dynamin在网格蛋白涂层的囊泡（CCV）形成中还具有至关重要的作用。目前，Dynamin被认为是一种分子“ Pinchase”，可促进质膜中CCV的萌芽，但是Schmid Lab的最新证据支持其作为内吞作用的关键调节剂的作用。因此，我们的目标是通过：l）定义元素募集到网状蛋白涂层坑所需的分子，并确定dymanin功能的上游和下游效应子。为了实现这些目标，我们将使用与功能相关的结构采用严格的生化方法，通过亲和力色谱筛选细胞提取物。发现与动力蛋白特别相互作用的因素将立即测试其使用无细胞的内吞作用的囊泡形成中离散步骤的能力，以忠实地重新构建了体外CCV的产生。网格蛋白介导的内吞途径是基本的，高度保守的，并且由于已经在人类疾病和开发中报道了某些内吞机械蛋白的异常表达或突变，因此从该项目获得的信息不仅会增加我们对克拉斯蛋白介导的内细胞增多症的基本机制的理解，而且可以为某些疾病提供洞察力类型。