Defining the structural organization of the polysaccharide-synthesizing multiprotein complexes localized in Golgi

该跨学科项目将揭示植物高尔基体多糖生物合成中涉及的生物合成机器的结构，并揭示它们的确切功能。木葡聚糖是大多数植物原代细胞壁的主要半纤维素成分，在植物系统的生长、发育、信号传导和胁迫反应中发挥着不可或缺的作用。这项工作应该适用于各种各样的植物物种，从而有助于基础研究和开发更理想的细胞壁结构，以实现健壮和多产的作物。总的来说，这些发现可以为提高作物质量和产量、增加粮食和能源安全和独立的生物技术战略奠定基础。该项目将为高中、本科和研究生阶段的初级科学家提供广泛的现代细胞和结构生物学和生物化学前沿研究方法的研究培训。通过这个项目的框架，研究生将发展指导和指导高中和本科生的教学技能。该项目的总体目标是通过实现三个具体目标来产生控制多糖模式组装的机制的知识：1)研究木糖基转移酶-底物相互作用，确定受体-底物结合模式；2)研究木糖基转移酶T异二聚体或异四聚体和木糖基转移酶- CSLC4配合物在高尔基体中的形成，阐明木葡聚糖合成配合物的结构组织；3)利用单粒子电子显微镜和计算模型对配合物及其动力学进行结构表征。提出的研究将通过在结构、生化和计算生物学方面的应用合作进行，包括活细胞成像、蛋白质免疫沉淀和酶分析、x射线晶体学、单颗粒EM和应用于多尺度系统的计算建模，如重组蛋白、植物和酵母细胞以及整个植物。该项目由分子和细胞生物科学部的细胞动力学和功能以及分子生物物理学小组共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This interdisciplinary project will uncover the structures of biosynthetic machines involved in polysaccharide biosynthesis in plant Golgi and unravel exactly how they function. Xyloglucan is the major hemicellulose component of primary cell walls in most plants and provides integral functions within plant systems for growth, development, signaling, and stress responses. The work should be applicable to a broad variety of plant species and hence contribute to both fundamental research and the development of more desirable cell wall structures for robust and productive crops. Overall, the findings could lay foundation for biotechnological strategies towards improving crop quality and yield increasing food and energy security and independence. The project will provide research training across a broad range of cutting-edge research methodologies in modern cell and structural biology and biochemistry for junior scientists at the high school, undergraduate, and graduate level. Graduate students will develop mentoring and teaching skills supervising high school and undergraduate students through the framework of this project. The overarching goal of this project to generate knowledge of mechanisms that control polysaccharide pattern assembly through the accomplishment of three specific aims: 1) Investigate xylosyltransferase - substrate interactions to confirm the acceptor substrate binding mode, 2) Investigate formation of xylosyltransferase T heterodimers or hetero-tetramers and xylosyltransferase - CSLC4 complexes in Golgi to elucidate the structural organization of the xyloglucan synthesizing complex, and 3) Structurally characterize the complexes and their dynamics using single particle EM and computational modeling. The research proposed will be carried out through a collaboration in applications across structural, biochemical, and computational biology through live cell imaging, protein immunoprecipitation and enzymatic assays, X-ray crystallography, single particle EM and computational modeling applied to multi-scale systems, such as recombinant proteins, plant and yeast cells and a whole plant. This project is jointly funded by the Cellular Dynamics and Function and Molecular Biophysics Clusters in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.