Probing the Energetic Cost of Cargo Encapsulation in Coated Vesicles

描述(申请人提供)：在突触传递和蛋白质运输等细胞过程中组装被覆膜囊泡(CV)需要跨膜货物分子和外壳蛋白之间的协调作用，迅速将膜塑造成包裹特定货物的高度弯曲的CV。病原体对CV的组装和利用的缺陷会导致毁灭性的疾病，每年累积影响数亿患者。虽然人们已经在很大程度上确定了血管的分子组成和结构，但关键的生理问题仍然没有答案。具体地说，确定外套的物理感觉和适应货物的方式，并阐明决定CV大小和货物含量的标准，是了解CV组装在人类疾病中的作用的关键剩余步骤。为了解决这些问题，拟议工作的目标是量化和比较货物包装的能量成本与涂层组件在CV形成过程中的能量贡献。我们实验室最近的工作表明，包裹货物分子的能量成本随着它们在膜表面的浓度呈指数增加，这是它们之间空间压力增加的结果。同样，我们的工作表明，将涂层组分浓缩到CV中的水平时，会在相反的膜表面产生巨大的立体压力，从而驱动膜的弯曲，而不是来自货物分子的压力。与目前的理解相反，这些结果表明，集中的货物分子，而不是弯曲的膜，是形成CVS的主要物理障碍。这些观察结果导致了一个中心假设，即外套晶格的组装立体地限制了膜的外壳和涂层侧的分子成分，在共同塑造新生CV的相反的膜表面压力之间建立了竞争。以笼状蛋白包被坑的组装为模型系统，三个目标的实验将验证这一假说。使用最小的膜系统和定量光学分析，目标1中的实验将测量货物封装的能量成本作为货物浓度和分子质量的函数。相比之下，AIM 2的实验将使用最小系统来量化和比较货物封装的能量驱动因素，包括涂层聚合、涂层组分之间的空间压力和疏水插入。最后，目标3将探索在活细胞中包裹货物的成本和驱动因素之间的生理平衡。这些实验将确定货物浓度和相对分子质量对CV的大小和毛皮组成的影响。使用创新的方法来量化CV形成的能量学，拟议工作的意义将是对货物和衣物成分之间的能量竞争决定CV的大小和分子含量的程度的关键评估，这是理解和解决由于CV组装的错误调节、突变和病原性利用而引起的病理的关键一步。

DESCRIPTION (provided by applicant): Assembling coated membrane vesicles (CVs) during cellular processes such as synaptic transmission and protein traffic requires coordinated interactions between transmembrane cargo molecules and coat proteins, rapidly shaping the membrane into a highly curved CV that encapsulates a specific cargo. Defects in CV assembly and exploitation of CVs by pathogens lead to devastating diseases that cumulatively impact hundreds of millions of patients each year. While the molecular components and structures of CVs have been largely identified, critical physiological questions remain unanswered. Specifically, determining how the coat physically senses and adapts to cargo and elucidating the criteria that determine the size and cargo content of CVs are key remaining steps toward understanding the role of CV assembly in human disease. To address these questions, the objective of the proposed work is to quantify and compare the energetic costs of cargo encapsulation with the energetic contributions of coat assembly during CV formation. Recent work in our lab has demonstrated that the energetic cost of encapsulating cargo molecules increases exponentially with their concentration on membrane surfaces, a consequence of increased steric pressure among them. Similarly, our work has shown that concentrating coat components to the levels found in CVs creates a substantial steric pressure on the opposite membrane surface that drives membrane curvature, in opposition to pressure from cargo molecules. In contrast to current understanding, these results suggest that concentrating cargo molecules, rather than bending membranes, represents the major physical barrier to forming CVs. These observations lead to the central hypothesis that assembly of the coat lattice sterically confines molecular components on the cargo and coat sides of the membrane, setting up a competition between opposing membrane surface pressures that collectively shape nascent CVs. Using assembly of clathrin-coated pits as a model system, experiments in three aims will test this hypothesis. Using minimal membrane systems and quantitative optical assays, experiments in Aim 1 will measure the energetic cost of cargo encapsulation as a function of cargo concentration and molecular mass. In contrast, experiments in Aim 2 will use minimal systems to quantify and compare the energetic drivers of cargo encapsulation, including coat polymerization, steric pressure among coat components, and hydrophobic insertion. Finally, Aim 3 will probe the physiological balance between the costs and drivers of cargo encapsulation in living cells. These experiments will determine the impact of cargo concentration and molecular weight on the size and coat composition of CVs. Using innovative methodologies to quantify the energetics of CV formation, the significance of the proposed work will be a critical evaluation of the extent to which an energetic competition between cargo and coat components determines the size and molecular content of CVs, a key step toward understanding and addressing pathologies arising from misregulation, mutation, and pathogenic exploitation of CV assembly.