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关键作用，它发生在质膜(PM)的特殊区域，称为被膜小孔。在这些位置，网状蛋白和AP组装成一个蛋白质晶格，该晶格浓缩了受体-配体复合体并使膜变形。然后，GTP酶动力蛋白在深深凹陷的涂层凹坑的颈部自组装成一个项圈，需要它来分离涂层小泡。除了这些内吞机制的主要成分外，还根据它们与笼蛋白、AP或Dynamin的相互作用鉴定了几种新的蛋白质。然而，我们不知道这些成分中的哪些构成了囊泡萌发的最基本的必要机制，它们的作用机制，或者辅助成分在这一过程中的作用。我建议开发新的分析方法，使用高纯度的PM组分，高效和忠实地重建CCV形成过程中的每一步，包括AP2、网状蛋白和动力素招募、配体隔离和内化以及游离包膜囊泡的释放。从长远来看，这些特定阶段的分析将被用来建立最低要求、分子相互作用的等级以及管理涂层囊泡形成的机制。

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.