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

动态 COPII 涂层组装驱动内质网囊泡形成的机制。

• 批准号: EP/X027295/1
• 负责人: Sarah Triclin
• 金额: $ 26万
• 依托单位: MRC Laboratory of Molecular Biology
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FX027295%2F1
• 关键词: Mechanisms; dynamic; COPII; coat; assembly

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.

蛋白质分泌是一个重要的过程，在所有真核生物中都是保守的，由携带新生分泌蛋白和膜蛋白的内质网（ER）萌发的copii包被囊泡启动。COPII囊泡由5个保守的“外壳”蛋白组成，这些蛋白自组装同时选择货物蛋白并将膜雕刻成囊泡载体。要做到这一点，涂层必须满足几个看似矛盾的原则：它必须(1)足够坚固，能够施加很大的力来弯曲膜；(2)本质上不稳定，允许剥离，从而驱动与高尔基体膜的融合和蛋白质/脂质传递；(3)具有结构适应性，使得运输载体的形态可以包含小囊泡（60-80纳米）以及携带前胶原蛋白（300纳米棒）或脂蛋白颗粒（300纳米球体）等货物的较大结构。了解COPII涂层组装途径如何被启动、调节和修改以产生具有不同形态的转运中间体是我项目的重点。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，并旨在提供新的方法来解决如何调节外壳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，以可视化支持双层上的涂层组装。这种重构将允许在生理相关状态的背景下分析被毛的动力学，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被毛突变体库将进一步揭示控制被毛组装和适应性的机制。