Functional Domains and Spatial-Temporal Interactions of an Arabidopsis Circadian Clock-Associated F-Box Protein

泛素蛋白水解是一种修饰和降解系统，对所有真核生物的正常功能至关重要。在cullin泛素连接酶中，SCF类是最多样化的，有近700个F-box蛋白被确定。ZEITLUPE （ZTL）是参与调节植物生物钟的关键F-box蛋白。最近对ZTL蛋白水解靶点（TOC1）的鉴定，将使人们能够专注于ZTL的功能解剖，其细胞内作用位点，并确定其他生化相互作用物。利用ZTL的缺失结构和新发现的点突变，将确定植物TOC1/ZTL相互作用所需的结构域和残基。通过实验控制和监测它们的区隔，还将确定ZTL/TOC的空间和时间相互作用。此外，将确定ztl介导降解的直接目标。参与原生SCFZTL复合体的特定ASK家族成员和其他额外的相互作用伙伴也将被研究。这项工作将为参与的研究生和博士后研究人员提供一个在互动环境中学习当代分子生物学和植物分子遗传技术的机会。此外，更有经验的实验室工作人员将指导本科生，因此他们将学习技术和分子技能，他们可以在以后的职业生涯中使用。其他影响包括昼夜节律生物学课程的发展和指导，旨在调查和综合所有昼夜节律系统的当前主题。本项目研究人员的科研经验将拓宽他们的科学专业知识，有助于他们未来成为PI、博士后和教师。

Ubiquitin-based proteolysis is a modification and degradation system that is essential to proper functioning of all eukaryotes. Of the cullin ubiquitin ligases, the SCF class is the most diverse, with nearly 700 F-box proteins identified. ZEITLUPE (ZTL) is a key F-box protein involved in regulating the plant circadian clock. The recent identification of a proteolytic target of ZTL (TOC1), will now allow focus on the functional dissection of ZTL, its intracellular site of action, and identify additional biochemical interactors. Using deletion constructs of ZTL, and newly identified point mutations, the domains and residues necessary for in planta TOC1/ZTL interactions will be identified. The spatial and temporal ZTL/TOC interaction will also be identified, by experimentally controlling and monitoring their compartmentation. Furthermore direct targets of ZTL-mediated degradation will be determined. The specific ASK family member(s) that participate in the native SCFZTL complex and other additional interacting partners will also be studied. This work will provide an opportunity for the graduate student and postdoctoral researchers involved to learn contemporary molecular biology and plant molecular genetic techniques in an interactive environment. Additionally, the more experienced lab workers will mentor undergraduates, so they will learn technical and molecular skills that they could use later in their career. Additional impacts include the development and instruction of a Circadian Biology course designed to survey and synthesize current topics in all circadian systems. The research experiences of the personnel in this project will broaden their scientific expertise, and contribute to their development as future PI's, post-docs and teachers.