Mechanisms of dynamic COPII coat assembly to drive vesicle formation from the endoplasmic reticulum.

蛋白质分泌是所有真核生物中保守的一个重要过程，由从内质网（ER）出芽的COPII包被的囊泡启动，携带新生分泌和膜蛋白。COPII囊泡由五种保守的“外壳”蛋白形成，这些蛋白自组装以同时选择货物蛋白并将膜雕刻成囊泡载体。为了实现这一点，包衣必须满足几个看似矛盾的原则：它必须（1）足够坚固，以施加显著的力来弯曲膜;（2）本质上不稳定，以允许脱包衣，这驱动与高尔基体膜的融合和蛋白质/脂质递送;以及（3）在结构上是可适应的，使得转运载体的形态可以包含小的囊泡（60-80 nm）以及携带货物的较大结构，如前胶原（300 nm棒）或脂蛋白颗粒（300 nm球）。了解COPII外套组装途径是如何启动，调节和修改，以产生不同形态的运输中间体是我的项目的重点。我建议研究COPII组装途径如何在动态蛋白质-蛋白质和蛋白质-脂质相互作用的背景下进行，并旨在带来新的方法来解决如何调节外套组装以适应特定生理需求的问题。我将开发一种实时成像分析，可视化支持双层上的涂层组装。这种重建将允许在生理相关状态的背景下分析外套的动力学，包括核苷酸（GTP），货物和辅助蛋白。此外，利用米勒实验室提供的特定外套突变体库将进一步揭示控制外套组装和适应性的机制。

Protein secretion is an essential process that is conserved across all eukaryotes and is initiated by COPII-coated vesicles that bud from the endoplasmic reticulum (ER) carrying nascent secretory and membrane proteins. COPII vesicles are formed by five conserved "coat" proteins that self-assemble to simultaneously select cargo proteins and sculpt the membrane into a vesicle carrier. To achieve this, the coat must fulfil several seemingly contradictory principles: it must be (1) sufficiently robust to exert significant force to bend the membrane; (2) intrinsically unstable to permit uncoating, which drives fusion with the Golgi membrane and protein/lipid delivery; and (3) be architecturally adaptable, such that the morphology of transport carriers can encompass small vesicles (60-80 nm) as well as larger structures that carry cargos like pro-collagen (300nm rods) or lipoprotein particles (300nm spheres). Understanding how the COPII coat assembly pathway is initiated, regulated, and modified to generate transport intermediates with diverse morphologies is the focus of my project. I propose to investigate how the COPII assembly pathway proceeds in the context of dynamic protein-protein and protein-lipid interactions, and aim to bring new methodologies to solve the problem of how coat assembly is modulated to adapt to specific physiological needs. I will develop a real-time imaging assay that visualizes coat assembly on supported bilayers. Such reconstitution will allow analysis of the dynamics of the coat in the context of physiologically relevant states, including nucleotide (GTP), cargo, and accessory proteins. Moreover, capitalizing on the library of specific coat mutants available in the Miller lab will further reveal mechanisms that govern coat assembly and adaptivity.