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

目的和方法：本研究项目的长期目标是了解内体在植物中的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，内体隔室在膜蛋白的分类、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起着重要的作用。PI和她的团队将研究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 ATPase。一个导致AtSKD1功能中断的可诱导表达系统将被用作研究植物内质体室的组织和功能特性的工具。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，结合荧光成像、金免疫标记和电子断层扫描。这些研究的具体目的是：(1)研究AtSKD1在植物内体和液泡系统结构组织中的功能。(2)分析AtSKD1如何调控不同的植物内体运输途径。(3)鉴定和分析AtSKD1相互作用的蛋白。智力价值：这些研究的完成将澄清植物内体系统的功能和结构组织，这在细胞生物学领域是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如何组织的信息，严重限制了对质膜蛋白、受体和生物合成货物运输机制的理解。有充分的理由相信，植物的内体系统比酵母和动物细胞中的内体系统更复杂和多样化。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。内体运输在植物生长发育、细胞类型确定、细胞壁重塑、向重力反应、信号转导和激素运输等方面发挥着重要作用。这里提出的研究将为这一植物细胞生物学领域提供有价值的信息。广泛的影响：该项目将对教育和研究产生影响。PI目前在威斯康星大学教授本科生课程。两名本科生已经把这项提案的一部分作为他们研究项目的中心话题。更多的本科生，优先来自代表人数不足的少数群体，将很快被纳入这一项目。新成像技术的发展对教育也将是重要的，因为PI致力于为校园内的本科生和研究生开发关于细胞生物学和细胞成像的研讨会，作为威斯康星大学植物学系新成像设施教育活动的一部分，该设施已得到NSF资金的部分支持。此外，在这项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。这些断层照片也将提供给教师。重要的是要注意，电子断层图像可以在使用完全自由软件(http://bio3d.colorado.edu).)的传统PC或Macintosh计算机上显示因此，经过适当培训的教师将能够使用电子断层扫描作为植物细胞生物学课程的宝贵工具，基本上是免费的。

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.