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质货物的膜结合囊泡和小管递送至 通过特定运输途径的离散细胞位置对于细胞生物学和人类健康至关重要。 许多与贩运途径有关的蛋白质与严重和致残的人类疾病有关， 包括阿尔茨海默氏症和遗传性痉挛性截瘫等神经系统疾病。虽然某些 运输蛋白质和途径的特点，我们缺乏直接的证据或只有部分证据 我们推断膜之间一定存在其他通路。这在我们的国家中构成了巨大的差距。 目前对基本细胞生物学的理解。 我们的目标是阐明重要的外壳蛋白复合物的分子结构和功能， 通过在特定膜上的囊泡或小管周围形成涂层来运输途径。外壳蛋白 识别和包装相关货物，它们促进额外所需蛋白质的有效组装 组件，如SNARE。虽然网格蛋白外壳已被广泛研究，但某些非网格蛋白的功能 大衣仍然几乎未知。越来越多的证据表明，非网格蛋白外套组装使用不同的 机制，表明网格蛋白不能被认为是一个范例的外套组装。我们调查非- 网格蛋白外壳复合物，包括衔接蛋白4（AP 4）、外壳蛋白复合物I（COPI）和逆转录聚合物， 使用各种工具来确定外套组装和调节的分子机制。生化和 蛋白质组学方法允许我们鉴定包被结构的新组分，特别是货物分子， 辅助蛋白和调节蛋白。结构方法，如X射线晶体学，NMR和电子 显微镜在分子水平上揭示了外套是如何与关键蛋白质伴侣相互作用的， 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，并探测 在结构模型中识别接口。与合作者，我们使用分子数据来设计实验， 培养的细胞系和模型生物，以探索各种蛋白质-蛋白质相互作用如何驱动 在细胞和生物体水平上的表型。最终，我们希望能够从分子水平上了解 非网格蛋白涂层如何在不同的膜上组装以驱动不同的运输途径。我们预计 这项工作将揭示货物识别，外套组装和调节的新机制。我们进一步的目标是 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.