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，发生在质膜（PM）的特定区域，称为被膜孔。 在这些位点，网格蛋白和AP组装成蛋白质晶格，其浓缩受体-配体复合物并使膜变形。 然后，GT3发动蛋白在深深内陷的包被小凹的颈部自组装成一个项圈，并需要分离包被囊泡。 除了内吞机制的这些主要成分外，还根据它们与网格蛋白、AP或发动蛋白的相互作用鉴定了几种新蛋白质。 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必要机制，它们的作用机制，或者辅助成分在这个过程中的功能。 我建议开发新的检测方法，使用高度纯化的PM组分，有效地和忠实地重建每一个步骤沿着CCV形成的途径，包括AP 2，网格蛋白，发动蛋白招聘，配体螯合和内化，释放自由包被囊泡。 从长远来看，这些阶段特异性试验将用于建立最低要求，分子相互作用的层次结构和包衣囊泡形成的机制。

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.