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

基于泛素的蛋白质降解是一种修饰和降解系统，对于所有真核生物的正常运作是必不可少的。在剔除泛素连接酶中，SCF类是最多样化的，有近700种F-box蛋白被鉴定。ZEITLUPE(ZTL)是一种关键的F-box蛋白，参与调节植物生物钟。最近发现了ZTL(TOC1)的一个蛋白水解靶，现在将可以专注于ZTL的功能解剖，它的细胞内作用部位，并确定其他生化相互作用。利用ZTL的缺失构建体和新发现的点突变，将确定在植物TOC1/ZTL相互作用中所需的结构域和残基。还将通过实验控制和监测ZTL/TOC的分隔来确定它们的空间和时间相互作用。此外，还将确定ZTL介导的降解的直接目标。还将研究参与原生SCFZTL情结的特定ASK家庭成员(S)和其他额外的互动伙伴。这项工作将为参与其中的研究生和博士后研究人员提供在互动环境中学习当代分子生物学和植物分子遗传学技术的机会。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，这些技能可以在以后的职业生涯中使用。其他影响包括开发和指导昼夜节律生物学课程，旨在调查和综合所有昼夜节律系统中的当前主题。该项目人员的研究经验将拓宽他们的科学专长，并为他们未来的PI、博士后和教师的发展做出贡献。

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.