植物ESCRT复合物组分蛋白的泛素化修饰及其调控膜蛋白转运和降解的分子机制研究

The endosomal sorting complex required for transport (ESCRT) machinery is mainly responsible for the regulation of multivesicular body (MVB) biogenesis as well as the MVB-mediated membrane protein sorting and degradation (such as the activated membrane receptors), and is widely involved in the regulation of plant development and environmental responses. However, the post-translational modifications of ESCRT components and the biological functions of these modifications are still lack of study in plants. This proposal intends to study the regulation and function of ubiquitin modification of FREE1, a plant unique ESCRT component, in the plant vesicular transport pathway. Our study includes the regulatory mechanism of FREE1 ubiquitination and ubiquitin-mediated degradation, the identification of FREE1 ubiquitination sites and its functional analysis, the functional roles of FREE1 ubiquitination in the assembly of ESCRT complexes as well as in the MVB-mediated memebrane protein sorting and degradation. We will focus on the regulatory mechanism of FREE1 ubiquitination and the functions of FREE1 ubiquitination in plant ESCRTing pathway. Eventually, we will clarify the molecular function of ubiquitination in the regulation of ESCRT-mediated membrane protein sorting and degradation. This study will reveal the regulatory mechanism and the functions of ubiquitination modifications of ESCRT components. Thus, this study can contribute to the research of ESCRTing machinery in eukaryotes, including the research in mammal and yeast.

内吞体分选转运复合物（ESCRT）主要负责调控多泡体（MVB）的形成及MVB介导的泛素化膜蛋白的分选和降解，因而广泛参与植物的生长发育和环境应答。然而植物中ESCRT复合物蛋白组分本身的翻译后修饰及其生物学功能尚缺乏相关研究。本项目拟以植物特有的ESCRT复合物组分蛋白FREE1的泛素化修饰调控及其功能研究为切入点，从分子细胞及遗传等多个角度研究FREE1蛋白泛素化修饰和降解的调控过程、FREE1蛋白的泛素化修饰位点及其功能分析、泛素化修饰调控ESCRT复合物的组装以及MVB介导的膜蛋白的分选和降解，重点研究FREE1蛋白泛素化修饰的调控机制和功能分析，最终阐明泛素化修饰调控ESCRT复合物功能及膜蛋白分选和降解的分子机制。本研究将揭示植物ESCRT复合物组分本身的翻译后修饰和功能的调控机制，为真核生物包括动物和酵母中ESCRT复合物的功能研究提供借鉴。

内吞体分选转运复合物（ESCRT）作为真核生命中保守的蛋白超复合体主要负责识别泛素化修饰的膜蛋白，以及调控多泡体（MVB）的形成和MVB介导的膜蛋白转运和降解。本实验室前期研究发现了一个植物特有的ESCRT复合物组分并将其命名为FREE1，解析了FREE1作为ESCRT组分调控MVB形成、液泡蛋白转运和液泡形成以及自噬降解的功能。虽然FREE1蛋白基本的生物学功能已经被研究地较为清楚，但是该蛋白本身的功能调控，尤其是蛋白的翻译后修饰及其蛋白稳定性调控，以及这些调控过程与植物逆境或生长信号的关联机制尚不清楚。本研究发现SINATs E3泛素连接酶调控FREE1蛋白的泛素化修饰和蛋白稳定性，解析了FREE1蛋白泛素化修饰响应和调控植物缺铁信号的分子机制。在缺铁环境下，SINAT-1、-2、-3和-4四个E3泛素连接酶能将FREE1蛋白泛素化修饰，并促进自噬相关基因ATGs的表达进而激活细胞自噬，最终促进FREE1蛋白的泛素化修饰和经由自噬途径降解，缓解植物缺铁的胁迫。而RING结构域缺失的SINAT5蛋白无E3泛素连接酶活性，并且可以抑制SINAT1对FREE1的泛素化修饰，保护FREE1免受泛素化修饰和降解。本研究首次发现了FREE1蛋白的翻译后修饰以及蛋白稳态调控机制，并揭示了ESCRT途径、自噬途径以及植物缺铁信号响应之间存在的潜在关联调控关系，丰富了植物缺铁信号调控网络。