Molecular mechanisms of endocytic initiation and cargo selection

摘要 从质膜内化的主要途径是网格蛋白介导的内吞作用（CME）， 是至关重要的细胞过程所必需的，如信号传导、营养吸收和发育。同时分子 囊泡形成的后期阶段的机制得到了很好的研究， 内吞作用的最早阶段，包括起始和货物选择，知之甚少。启动和货物 结合在很大程度上由AP 2网格蛋白衔接子复合物控制，AP 2网格蛋白衔接子复合物是囊泡外壳的核心组分， 作为刚性网格蛋白晶格和膜包埋货物之间的桥梁。然而，目前还不清楚如何 AP 2在复杂的膜环境中区分数百种潜在的货物，同时还 对空间和时间的调节线索作出反应。虽然超过50种蛋白质被提议调节或需要 对于CME，以及许多与AP 2物理相互作用的物质，我们几乎没有关于它们如何与AP 2相互作用的机制和结构数据。 在胞吞作用的最早阶段受到调节。我们提出，多个独特的AP 2构象，驱动 通过与调节因子的关联，控制高阶AP 2功能，如货物选择。理解 控制内吞作用的调节机制的性质是至关重要的，因为CME在很大程度上控制着细胞内吞作用。 许多医学相关蛋白质的定位，例如RTK和GPCR。因此，这项建议旨在 重建和定义AP 2介导的内吞起始和货物分选的分子机制。 目前的内吞作用模型主要依赖于用可溶性组分进行的生化实验， 活细胞成像重要的是，我们的方法集中在修改所有的实验方法-冷冻EM， 生物化学重建和单分子荧光显微镜-包括膜，从而 解决了在近天然膜环境中开发机械模型的关键需求。这 这种方法准备提供一个理解的作用，膜诱导的变构在驱动调节 在内吞起始期间的决策。这项建议将集中在两个广泛的领域内吞调节 - 由保守的Muniscin家族蛋白介导的货物选择，以及质量控制检查点 由AP 2的μ2亚基上的单个磷酸化标记控制。由于与内吞相关的疾病 缺陷可能是由重要的跨膜货物的错误分类引起的，我们对 内吞起始和货物选择将使假说驱动的研究进入疾病模型系统， 药物开发

Abstract The primary route of internalization from the plasma membrane is clathrin-mediated endocytosis (CME), which is required for vital cellular processes such as signaling, nutrient uptake, and development. While the molecular mechanisms underlying the late stages of vesicle formation are well studied, the mechanisms underpinning the earliest stages of endocytosis, including initiation and cargo selection, are poorly understood. Initiation and cargo binding are largely controlled by the AP2 clathrin adaptor complex, a core component of the vesicle coat that serves as a bridge between the rigid clathrin lattice and membrane-embedded cargo. However, it is unclear how AP2 discriminates between hundreds of potential cargo in a complex membrane environment, while also responding to spatial and temporal regulatory cues. While >50 proteins are proposed to regulate or be required for CME, and many physically interact with AP2, we have little mechanistic and structural data for how they are regulated during the earliest stages of endocytosis. We propose that multiple unique AP2 conformations, driven by association with regulatory factors, control higher-order AP2 functions such as cargo selection. Understanding the nature of the regulatory mechanisms controlling endocytosis is critical, as CME largely controls the localization of many medically-relevant proteins such as RTKs and GPCRs. This proposal therefore seeks to reconstitute and define the molecular mechanisms of AP2-mediated endocytic initiation and cargo sorting. Current models of endocytosis largely rely on biochemical experiments performed with soluble components and live cell imaging. Importantly, our methodology is focused on modifying all experimental approaches — cryo-EM, biochemical reconstitution, and single molecule fluorescence microscopy — to include a membrane, thereby addressing a critical need to develop mechanistic models in a near-native membrane environment. This approach is poised to provide an understanding of the role of membrane-induced allostery in driving regulatory decision-making during endocytic initiation. This proposal will focus on two broad areas of endocytic regulation — cargo selection mediated by the conserved Muniscin family proteins, and a quality control checkpoint controlled by a single phosphorylation mark on the μ2 subunit of AP2. As diseases associated with endocytic defects are likely caused by missorting of important trans-membrane cargo, our insights into the mechanisms of endocytic initiation and cargo selection will enable hypothesis-driven research into disease model systems and drug development.