MOLECULAR MECHANISM OF CLATHRIN COATED VESICLE FORMATION

受体介导的内吞作用在许多重要的生理过程中起着至关重要的作用，发生在质膜（PM）的特殊区域，称为被涂坑。在这些位点上，网格蛋白和ap聚集成一个蛋白质晶格，使受体-配体复合物集中，使膜变形。然后，GTPase动力蛋白在深内陷的包覆凹坑的颈部自组装成一个领子，并需要分离包覆的囊泡。除了这些内吞机制的主要成分外，基于它们与网格蛋白，APs或动力蛋白的相互作用，已经确定了几种新的蛋白质。然而，我们不知道这些成分中的哪一个构成了囊泡出芽的最小基本机制，它们的作用机制，或辅助成分在这一过程中的功能。我建议开发一种新的检测方法，使用高度纯化的PM组分，有效而真实地重建CCV形成途径的每一步，包括AP2、网格蛋白和动力蛋白的募集、配体的隔离和内化，以及自由包被囊泡的释放。从长远来看，这些阶段特异性分析将用于建立最低要求，分子相互作用的层次结构，以及控制包被囊泡形成的机制。

Receptor-mediated endocytosis, which plays a critical role in many physiologically important processes, occurs at specialized regions of the plasma membrane (PM), called coated pits. At these sites, clathrin and APs assemble into a protein lattice that concentrates receptor-ligand complexes and deforms the membrane. The GTPase dynamin then self-assembles into a collar at the necks of deeply invaginated coated pits and is required to detach a coated vesicle. In addition to these major constituents of the endocytic machinery, several new proteins have been identified based on their interactions with clathrin, APs, or dynamin. However, we do not know which of these components constitutes the minimal essential machinery for vesicle budding, their mechanism of action, or the function of ancillary components in this process. I propose to develop novel assays using highly purified PM fractions that efficiently and faithfully reconstitute each step along the pathway of CCV formation, including AP2, clathrin, and dynamin recruitment, ligand sequestration and internalization, and the release of free coated vesicles. In the long-term, these stage-specific assays will be used to establish the minimal requirements, the hierarchy of molecular interactions, and the mechanisms governing coated vesicle formation.