Molecular mechanisms of coat assembly and regulation in membrane trafficking pathways

项目摘要 及时将携带跨膜蛋白和脂类货物的膜结合囊泡和小管运送到 通过特定的运输途径进行离散的细胞定位是细胞生物学和人类健康的基础。 许多与运输途径相关的蛋白质与严重的人类疾病有关， 包括阿尔茨海默氏症和遗传性痉挛截瘫等神经系统疾病。虽然可以肯定 运输蛋白和途径有很好的特征，我们缺乏直接证据或只有部分证据 因为我们推断的其他途径一定存在于膜之间。这构成了我们的巨大差距 目前对基础细胞生物学的理解。 我们的目标是阐明重要的外壳蛋白复合体的分子结构和功能 通过在特定膜上的小泡或小管周围形成涂层来运输的途径。外壳蛋白 识别和包装相关的货物，并促进额外所需蛋白质的有效组装 组件，比如陷阱。虽然网状蛋白涂层已被广泛研究，但某些非网状蛋白的功能 大衣几乎还不为人所知。越来越多的证据表明，非网格蛋白涂层使用DISTINCT进行组装 机制，这表明笼状蛋白不能被认为是涂层组装的范例。我们调查的不是 包括适配器蛋白4(AP4)、外壳蛋白复合体I(COPI)和逆转聚体在内的笼状蛋白外壳复合体，通过 使用各种工具来确定涂层组装和调节的分子机制。生化和 蛋白质组学方法使我们能够识别涂层结构的新成分，特别是货物分子， 附属物和调节蛋白。X射线结晶学、核磁共振和电子等结构方法 显微镜在分子水平上揭示了外壳如何与关键蛋白质伙伴相互作用，并使我们能够绘制 特定的绑定接口。生物物理技术使我们能够量化结合亲和力并探索 在结构模型中标识的界面。与合作者一起，我们使用分子数据来设计实验 在培养的细胞系和模型生物中探索各种蛋白质-蛋白质相互作用是如何推动 在细胞和生物水平上的表型。最终，我们希望从分子水平上了解 非网状蛋白涂层如何聚集在不同的膜上，以驱动不同的运输路径。我们期待着 这项工作将揭示货物识别、外套组装和调节的新机制。我们的进一步目标是 揭示与这些外壳蛋白相关的特定疾病的分子基础。

Project Summary The timely delivery of membrane-bound vesicles and tubules bearing transmembrane protein and lipid cargo to discrete cellular locations via specific trafficking pathways is fundamental to cell biology and human health. Many proteins associated with trafficking pathways are linked to serious and crippling human diseases, including neurological disorders like Alzheimer's and the hereditary spastic paraplegias. Although certain trafficking proteins and pathways are well characterized, we lack direct evidence or have only partial evidence for other pathways that we infer must exist between membranes. This constitutes an enormous gap in our current understanding of fundamental cell biology. Our goal is to elucidate the molecular structures and functions of important coat protein complexes that initiate trafficking pathways by forming coats around vesicles or tubules at specific membranes. Coat proteins recognize and package relevant cargoes, and they promote efficient assembly of additional required protein components, like SNAREs. While clathrin coats have been extensively studied, functions of certain non-clathrin coats remain virtually unknown. Increasing evidence indicates non-clathrin coats assemble using distinct mechanisms, suggesting clathrin cannot be considered a paradigm for coat assembly. We investigate non- clathrin coat complexes, including adaptor protein 4 (AP4), coat protein complex I (COPI), and retromer, by using a variety of tools to ascertain molecular mechanisms of coat assembly and regulation. Biochemical and proteomic approaches allow us to identify new components of coated structures, especially cargo molecules, accessory, and regulatory proteins. Structural methods like X-ray crystallography, NMR, and electron microscopy reveal at the molecular level how coats interact with key protein partners and allow us to map specific binding interfaces. Biophysical techniques enable us to quantify binding affinities and to probe interfaces identified in structural models. With collaborators, we use molecular data to design experiments in cultured cell lines and in model organisms to explore how a variety of protein-protein interactions drive phenotypes at the cellular and organismal levels. Ultimately, we hope to gain a molecular understanding of how non-clathrin coats assemble at distinct membranes to drive different trafficking pathways. We anticipate this work will reveal new mechanisms of cargo recognition, coat assembly, and regulation. We further aim to uncover the molecular basis of specific diseases associated with these coat proteins.