Molecular mechanisms of endocytic initiation and cargo selection

摘要 从质膜内化的主要途径是笼蛋白介导的内吞作用(CME)，这是 是重要的细胞过程所必需的，如信号、营养吸收和发育。而分子 对囊泡形成后期的潜在机制进行了很好的研究，支持 内吞作用的早期阶段，包括起始和货物选择，人们知之甚少。启运和装运 结合在很大程度上是由AP2网状蛋白适配器复合体控制的，它是囊泡外壳的核心成分， 作为坚硬的笼状晶格和膜嵌入货物之间的桥梁。然而，目前还不清楚是如何 AP2在复杂的膜环境中区分数百种潜在的货物，同时还 对空间和时间调节线索作出反应。而&gt；50蛋白质被建议调节或被要求 对于CME，以及许多与AP2进行物理交互的人，我们几乎没有关于它们是如何的机械和结构数据 在内吞作用的最早阶段被调节。我们提出了多种独特的AP2构象，驱动 通过与调节因子相关联，控制更高阶的AP2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 许多与医学相关的蛋白质，如RTK和GPCRs的定位。因此，这项建议旨在 重组并确定AP2介导的内吞起始和货物分拣的分子机制。 目前的内吞作用模型在很大程度上依赖于用可溶性成分和 活细胞成像。重要的是，我们的方法专注于修改所有的实验方法-冷冻-EM， 生化重组和单分子荧光显微镜--包括膜，从而 解决在接近原生的膜环境中开发机械模型的迫切需要。这 该方法准备提供对膜诱导变构在驱动调节中的作用的理解 内吞启动过程中的决策。这项提案将集中在两个广泛的内吞调节领域。 -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 受AP2的μ2亚基上的单个磷酸化标记控制。作为与内吞有关的疾病 缺陷可能是由于重要的跨膜货物分拣错误造成的，我们对 内吞启动和货物选择将使对疾病模型系统和 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.