Functional Analysis of AtSKD1 and AtSKD1-Interacting Proteins in the Organization of the Endosomal Pathway in Arabidopsis

目标和方法：本研究项目的长期目标是了解植物内体运输途径和调节内体分选过程的分子机制。在所有真核细胞中，内体区室在膜蛋白的分选、再循环和周转、受体的下调以及蛋白质向空泡和溶酶体的运输中发挥重要作用。PI及其团队将研究AtSKD1在植物内体运输途径中的功能。AtSKD1是酵母Vps4p和哺乳动物SKD1的同源物，它们是内体分选和运输所需的AAA ATP酶。一个诱导型表达系统，导致AtSKD1功能的中断将被用作一种工具来研究植物内体隔室的组织和功能特性。此外，还将鉴定和研究与AtSKD1相互作用的蛋白质。由于其复杂性，植物内体运输途径的研究将需要一个多学科的方法，因此，本项目涉及使用生物化学和分子生物学技术与荧光成像，金免疫标记，和电子断层扫描。具体目的是：（1）研究AtSKD 1在植物内体和液泡系统结构组织中的功能。(2)分析AtSKD1如何调节不同的植物内体运输途径。(3)鉴定和分析AtSKD1相互作用蛋白质。智力价值：这些研究的完成将阐明植物内体系统的功能和结构组织，这是细胞生物学领域中一个很大程度上未知的领域。事实上，缺乏关于植物内体系统是如何组织的信息正在对质膜蛋白、受体和生物合成货物的运输机制的理解施加严重限制。有充分的理由相信植物内体系统比酵母和动物细胞内体区室更复杂和多样。事实上，植物细胞具有独特的、多种类型的液泡和蛋白质运输途径。内体运输在植物生命的各个方面发挥作用，例如发育、细胞类型特化、细胞壁重塑、向重力反应、信号转导和激素运输。本研究将为植物细胞生物学领域提供有价值的信息。更广泛的影响：本项目将对教育和研究产生影响。PI目前在威斯康星州大学教授本科课程。两名本科生已经将这一建议的一部分作为他们研究项目的中心课题。更多的本科生，特别是来自代表性不足的少数群体的本科生，将很快被纳入这个项目。新的成像技术的发展也将是重要的教育，因为PI致力于开发细胞生物学和细胞成像的本科生和研究生在校园的新成像设施在植物系的教育活动的一部分，威斯康星州大学，这已部分由NSF基金支持研讨会。 此外，本研究中获得的所有断层图像将免费提供给其他研究人员。这将使该领域的其他科学家能够进一步分析我们重建的细胞体积中包含的植物内体或其他细胞器和结构。这些断层扫描图也将提供给教师。值得注意的是，电子断层图像可以使用IMOD（一种完全免费的软件）（http：//www.example.com）在传统PC或Macintosh计算机中显示。bio3d.colorado.edu因此，经过适当培训的教师将能够使用电子断层图像作为植物细胞生物学课程的宝贵工具，基本上没有成本。

Objectives and methods: The long term goal of this research project is to understand theendosomal trafficking pathways in plants and the molecular mechanisms regulating the endosomal sorting process. Endosomal compartments play a fundamental role in the sorting, recycling, and turnover of membrane proteins, the downregulation of receptors, and the trafficking of proteins to vacuoles and lysosomes in all eukaryotic cells. The PI and her group will study the function of AtSKD1 in the plant endosomal trafficking pathways. AtSKD1 is a homologue of yeast Vps4p and mammalian SKD1, which are AAA ATPases required for endosomal sorting and trafficking. An inducible expression system that causes the disruption of AtSKD1 function will be used as a tool to study the organization and functional properties of the plant endosomal compartment. In addition, proteins interacting with AtSKD1 will be identified and studied. Due to its complexity, the study of plant endosomal trafficking pathways will require a multidisciplinary approach and therefore, the present project involves the use of biochemical and molecular biology techniques integrated with fluorescent imaging, gold immunolabeling, and electron tomography. The specific aims are:(1) Study the function of AtSKD1 in the structural organization of the endosomal and vacuolar systems in plants.(2) Analyze how AtSKD1 regulates different plant endosomal trafficking pathways.(3) Identify and analyze AtSKD1-interacting proteins.Intellectual merit: The completion of these studies will clarify the functional and structuralorganization of the plant endosome system, an area that is largely unknown in the cell biology field. In fact, the lack of information on how the plant endosomal system is organized is imposing serious limitations in the understanding of trafficking mechanisms of plasma membrane proteins, receptors, and biosynthetic cargo. There are good reasons to believe that the plant endosomal system is more complex and diverse that the endosomal compartment in yeast and animal cells. In fact, plant cells posses unique, multiple types of vacuoles and protein trafficking pathways. Endosomal trafficking plays a role in various aspects of plant life, such as development, cell type specification, cell wall remodeling, gravitropic response, signal transduction, and hormone transport. The researchproposed here will provide valuable information in this plant cell biology area.Broader impacts: The project will have impact on education and research. The PI is currently teaching undergraduate courses in the University of Wisconsin. Two undergraduate students are already taking parts of this proposal as the central topic of their research projects. More undergraduates, preferentially from underrepresented minority groups, will be soon incorporated into this project. The development of new imaging techniques will be also important for education, since the PI is committed to develop workshops on cell biology and cell imaging for undergraduate and graduate students across campus as part of the educational activities of the new Imaging Facility in the Department of Botany-University of Wisconsin, which has been partially supported by NSF funds. In addition, all the tomograms obtained in this study will be freely available to other researchers. This will allow other scientists in the field to further analyze plant endosomes or other organelles andstructures contained in our reconstructed cellular volumes. These tomograms will also be available for teachers. It is important to note that electron tomograms can be displayed in conventional PCs or Macintosh computers using IMOD, a completely free software (http://bio3d.colorado.edu). Therefore, teachers with the appropriate training will be able to use electron tomograms as a valuable tool for plant cell biology courses at basically no cost.