Tonoplast Protein Trafficking in Arabidopsis

本研究的长期目标是确定和表征植物液泡-张力质体膜蛋白的转运机制。以前发现了张力质体蛋白运输的化学抑制剂和张力质体蛋白异常定位的突变体。生物活性探针表明，多种途径，包括一种新的高尔基非依赖性途径，可靶向液泡膜蛋白。该项目将确定膜蛋白靶向液泡的机制，以及这些机制如何促进液泡的生物发生。该工作计划将验证高尔基非依赖性途径参与细胞质蛋白运输的假设，并将研究磷酸肌醇脂在植物液泡生物发生中的新作用。本研究的具体目的是：1)通过对两种细胞质蛋白定位缺陷突变体的表征，确定受损的细胞质转运4 （ITT4）和ITT6基因在细胞质蛋白靶向中的作用。2)确定磷酸肌醇脂在液泡生物形成中的作用。3)利用活细胞实时显微技术表征抗质体蛋白靶向高尔基非依赖性通路的动力学特性。鉴于液泡膜蛋白在调节细胞膨胀中的重要作用，以及液泡的储存潜力，这些结果可能对未来植物环境胁迫响应生物学及其在农业上的应用具有重要影响。更广泛的影响本项目采用的化学和基因筛选将产生新的化学物质和突变体，使该领域受益，这些化学物质和突变体将可供科学界使用。本计画的部分实验适合高中及大学的研究活动，并将促进学生在分子生物学及高分辨活细胞荧光成像的最新技术方面的训练。PI将利用这些活动继续吸引来自北卡罗来纳大学科学与数学学院的少数民族高中生，目的是提高少数民族学生在大学攻读科学学位的兴趣。PI的实验室亦会接待橡树河中学的学生参观，以激发学生对科学相关领域的兴趣，并向公众传达科学研究的影响。

INTELLECTUAL MERITThe long-term goal of this research is to identify and characterize the mechanisms for trafficking of plant vacuole-tonoplast membrane proteins. Chemical inhibitors of tonoplast protein trafficking and mutants with abnormal localization of a tonoplast protein were previously identified. The bioactive probes indicated that multiple pathways, including a novel Golgi-independent pathway, target vacuole-membrane proteins. This project will identify the mechanisms by which membrane proteins are targeted to the vacuole, and how these contribute to its biogenesis. The work plan will test the hypothesis that a Golgi-independent pathway engages in tonoplast protein trafficking, and will investigate a novel role for phosphoinositide lipids in plant vacuole biogenesis. The specific aims for this research are: 1) To identify the roles of IMPAIRED TONOPLAST TRAFFICKING4 (ITT4) and ITT6 genes in tonoplast-protein targeting by characterization of two mutants with defects in tonoplast protein localization. 2) To determine the role of phosphoinositide lipids in vacuole biogenesis. 3) To characterize the dynamics of the Golgi-independent pathway for tonoplast-protein targeting using real-time microscopy of live cells. Given the essential roles of vacuole membrane proteins in regulating cell turgor, and the storage potential of the vacuole, these results may have future impacts on the biology of environmental stress responses in plants and its applications to agriculture. BROADER IMPACTSChemical and genetic screens employed in this project will benefit the field by generating novel chemicals and mutants that will be available to the scientific community. Some of the experiments in this project are amenable for high school and undergraduate research activities, and will promote the training of students in state-of-the-art techniques in molecular biology and fluorescent high resolution live-cell imaging. The PI will use these activities to continue to engage minority high school students from the NC School of Science and Mathematics with the goal of increasing the interest of minority students in pursuing science degrees within the University. The PI's laboratory will also host visits from River Oaks Middle School students to spark the interest of these students towards science-related fields, and to convey the impacts of scientific research to the public.