FREE1 复合体在植物自噬途径中的功能研究

Membrane proteins destined for degradation are usually ubiquitinated and sorted into the intra-luminal vesicles (ILVs) of PVCs/MVBs for subsequent degradation in vacuole/lysosome upon MVB-vacuole fusion. This process is facilitated by the endosomal sorting complex required for transport (ESCRT) machinery. We have recently identified and characterized a unique plant ESCRT component called FYVE domain protein required for endosomal sorting 1 (FREE1). Arabidopsis free1 mutant is seedling lethal and defective in the formation of ILVs in MVBs. Consequently, endocytosed plasma membrane (PM) proteins destined for degradation cannot reach the lumen of the vacuole and mislocalize to the tonoplast. In addition, FREE1 regulates vacuolar trafficking, vacuole biogenesis and autophagosome degradation in plants, whereras the crosstalk between the secretory and autophagic pathways is likely achieved via interaction with the autophagic regulator SH3P2. Thus, we have provided that first functional characterization of a plant FYVE domain protein, playing multiple functional roles in regulating MVB biogenesis, vacuolar sorting, vacuole biogenesis and autophagosome turnover in plants. However, the underlying mechnisms remain ellusive. Therefore, the central goal of this proposal is to illustrate the molecular mechanisms of FREE1 function in plants using a combination of biochemical, cellular, molecular and genetic approaches. Specific objectives include: 1) To identify and characterize the FREE1-interacting protein complex in plants; and 2) To study the roles of FREE1 complex in autophagic degradation in plants.

内体蛋白分选转运装置(Endosomal Sorting Complex Required for Transport，ESCRT)识别挑选膜蛋白进入多囊泡体的内部腔囊泡并将其运输到液泡降解。我们前期研究鉴定了一个独特的植物ESCRT组分 FYVE domain protein required for endosomal sorting 1 (FREE1)可以调控内部腔囊泡在多囊泡体里的形成，并影响胞吞质膜蛋白质到液泡的降解。FREE1也可以调节液泡运输以及形成，自噬体降解， 并且与自噬调控蛋白SH3P2之间相互作用影响分泌途径和自噬途径之间的交叉调控过程。 我们的研究第一次阐述了植物中FYVE结构域蛋白的多功能特性，但是其分子调控机制仍不清楚。该研究计划通过多种研究方法（生化，细胞，分子和遗传等）鉴定FREE1复合体组分并研究其在自噬途径中的功能与分子调控机制。

植物内体分拣转运复合体(ESCRT)在分拣蛋白，特别是膜蛋白通过多囊泡体(MVB)去液泡降解起到非常重要的作用。我们之前发现FYVE domain protein required for endosomal sorting1 (FREE1)是ESCRT复合体成员之一，正向调节MVB形成。然而FREE1和ESCRT复合体是怎样被调控的并不清楚。本实验室项目研究我们通过结合抑制因数遗传筛选，细胞和生化等多种研究方法，首次发现了一个植物特异的含有Bro1结构域的负向调控MVB形成的蛋白BRAF。BRAF和FREE1互相竞争结合ESCRT-1组分Vps23，动态调节FREE1在MVB膜上的聚集，从而调控MVB生成和膜蛋白的液泡运输。该成果是代表了在植物中ESCRT复合体调节MVB和植物液泡生成的全新机制方面的重大突破。 在自噬机理方面研究中，我们也是首次发现了Sec62蛋白转运复合体在衣霉素诱导的内质网（ER）胁迫下和自噬体标记蛋白ATG8共定位，说明该复合体介导了因内质网胁迫而发生的错误折叠蛋白通过自噬途径去液泡。我们还另外发现了选择性自噬的受体AtNBR1通过特异识别靶蛋白AtExo70E2来介导该蛋白和Exocyst复合体阳性细胞器EXPO的液泡降解。总的来说，我们的本项目的研究对了解植物蛋白通过自噬途径降解以及多囊泡体和液泡的生成提供了有着重要的指导意义新的见解和认识。