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

基于泛素的蛋白质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，鉴定了近700种F盒蛋白。ZEITLUPE（ZTL）是一种参与调节植物生物钟的关键F-box蛋白。最近确定的ZTL（TOC 1）的蛋白水解靶点，现在将允许集中在ZTL的功能解剖，其细胞内的作用位点，并确定额外的生化相互作用。使用ZTL的缺失构建体和新鉴定的点突变，将鉴定植物中TOC 1/ZTL相互作用所必需的结构域和残基。空间和时间ZTL/TOC的相互作用也将被确定，通过实验控制和监测其分隔。此外，将确定ZTL介导的降解的直接靶标。还将研究参与天然SCFZTL复合物和其他其他相互作用伴侣的特定ASK家族成员。 这项工作将为研究生和博士后研究人员提供一个机会，在互动的环境中学习当代分子生物学和植物分子遗传技术。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开发和教学，旨在调查和综合所有昼夜节律系统中的当前主题。在这个项目中的人员的研究经验将扩大他们的科学专业知识，并有助于他们作为未来的PI的，博士后和教师的发展。

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.