植物细胞器动态互作网络的建立：EXPO分泌体，自噬体和液泡

All eukaryotic cells contain distinct membrane-bounded compartments, including endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), the multivesicular body (MVB) or prevacuolar compartment (PVC) and vacuole or lysosome. A variety of extracellular macromolecules are internalized by endocytosis, a process involving multiple organelles such as early or recycling endosomes, late endosomes and vacuoles or lysosomes. On the other hand, exocytosis, including conventional (ER/Golgi-dependent) and unconventional (ER/Golgi-independent) protein secretion pathways, involves the delivery of secretory vesicles to the cell membrane for secretion of cellular contents (e.g. growth factors and cell wall enzymes) into the extracellular environment upon membrane fusion. As the largest membrane-bound plant-unique organelle, the vacuole serves as the primary catabolic compartment in plant cells, which contains acidic hydrolytic enzymes for degradation and recycling of various kinds of biomolecules. Vacuole is also the food storage center during plant growth and development. Normally, materials are secreted and delivered into the vacuole via membrane-bound compartments for degradation. Two major pathways are responsible for the sequestration of cargos into the plant vacuole: the MVB/PVC-mediated and the autophagosome-mediated degradation pathways. In recent years, we have made several novel and important findings (published or unpublished) that are unique to plant MVB-mediated and autophagosome-mediated vacuolar degradation and unconventional protein secretion pathways. Major findings include: 1) Using the Arabidopsis Exo70E2 (AtExo70E2) as a marker for exocytosis, we discovered a novel organelle termed EXPO (Exocyst-positive Organelle) and a new unconventional protein secretion (UPS) pathway unique in plants, as well as the plant-specific AtExo70E2-recruited exocyst complex for EXPO; 2) We have uncovered a plant-specific FYVE domain protein, named as FREE1, which is essential for MVB biogenesis and vacuolar sorting of membrane proteins, as well as provided the first evidence for crosstalk between MVB and autophagosome; 3) We have discovered that unique COPII coat proteins exhibit distinct subcellular localizations and different effects on ER cargo export in Arabidopsis; 4) Using live cell confocal imaging and 3D Electron Tomography analysis, we have demonstrated that the Arabidopsis ATG9 plays a unique role in plant autophagosome formation from the ER; 5) We have demonstrated that MON1-CCZ1 complex-mediated Rab7 activation is essential for PVC-to-vacuole trafficking in plants; and 6) We have recently established a new vacuole biogenesis model using whole cell 3D Electron Tomography technique. However, our understanding of the organelle interaction network in these pathways and how they contribute to plant growth and development remain fragmented. Therefore, the central goal of this proposal is to illustrate the interaction network and regulation mechanisms between the vacuole and other organelles in plant cells using a combination of 3D Electron Tomography, biochemical, cellular, molecular and genetic approaches. Specific objectives include: 1) To uncover and characterize the regulation mechanisms of interaction between MVB, provacuole and vacuole; 2) To identify and characterize the protein complex in mediating the autophagosome-vacuole pathway in plants; and 3) To illustrate the underlying mechanisms of EXPO interaction with PM and the role of vacuole in controlling EXPO homeostasis.

液泡是植物具有多种功能的最大细胞器，可以调节细胞渗透压维持细胞微环境，是物质代谢储藏中心。近年来我们对植物细胞器的研究取得了一系列成果：1）发现EXPO新型分泌体与细胞质膜融合参与植物非常规的蛋白分泌途径; 2）发现植物特异蛋白FREE1对MVB的生物发生和膜蛋白到液泡降解至关重要，并首次证明MVB和自噬体之间的互作; 3）发现独特的COPII蛋白通过特异配对调控蛋白质从ER的输出；4）首次鉴定植物自噬体形成过程并利用3D电子断层扫描重构技术证明其与ER 存在直接互作；5）发现Mon1-Ccz1蛋白复合体调控Rab7活性参与液泡运输；6）首次建立植物根系全细胞鉴定液泡生物发生。本项目旨在结合3D电子断层扫描重构，体外重组，生物化学，细胞分子和遗传学等多学科的方法，建立系统性的植物细胞器（EXPO，自噬体与液泡）的动态互作网络，为将来进一步研究它们在植物发育和生长过程的生理功能提供新思路。

我们之前在植物特有的多泡体(MVB)介导的液泡降解和自噬体介导的蛋白质分泌途径方面取得了新的重要发现。在本项目中，我们旨在利用三维电子断层扫描（ET）、生物化学、细胞、分子和遗传学的结合方法阐明植物细胞中液泡和其他细胞器之间的互作网络和调控机制。我们的新发现和主要贡献包括: 1)在MVB-液泡相互作用方面，我们在拟南芥根细胞和气孔细胞中建立了第一个具有纳米分辨率的MVB-液泡生物发生的全细胞ET模型；2)在内膜-自噬相互中，我们首次证明了Sec62是内质网-吞噬的选择性自噬受体，并阐明了内质网-吞噬调节盐胁迫诱导的未折叠蛋白反应（UPR）的机制；3)我们还证明了选择性自噬受体AtNBR1特异性识别和介导AtExo70E2和EXPO在植物中的液泡降解；4)在植物逆境响应的细胞机制中，我们确定了两个ER衍生的外壳蛋白复合物(COPII)II囊泡在植物非生物胁迫下调节自噬和自噬体形成的不同功能和机制；5)阐明了植物特有的ESCRT成分FREE1在调节植物萌发幼苗脂滴降解和胁迫条件下自噬体关闭中的多种功能和机制；6) 研究了VAMP724/726和ORP2A在植物自噬过程中调控自噬体形成和内质网-自噬体膜接触位点(MCS)的作用。综上所述，我们对了解植物细胞器网络及其功能做出了重要贡献。