Plant Vacuole Biogenesis and Function

植物液泡的生物发生和功能

• 批准号: 0212013
• 负责人: John Rogers
• 金额: --
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212013&HistoricalAwards=false
• 关键词: Plant; Vacuole; Biogenesis; Function

Plant cells store a diverse variety of molecules in vacuoles. Storage is the central reason why plants are so important to humankind. The wars against cocaine and heroin result from the harvest of products in storage vacuoles, the morning coffee is brewed to release products stored in vacuoles, the flowers in our garden are there because of pigments stored in vacuoles, and the nutrition of domestic animals and humans ultimately depends upon proteins stored in plant vacuoles. Many molecules that are targets in plant biotechnology are stored in vacuoles. Only by understanding the different compartments, their internal contents and environment, and how proteins and membrane are directed to each, will we be able to program a cell to make and accumulate a desired product. The need to separate a digestive compartment similar to the yeast vacuole or mammalian lysosome from the storage compartments has resulted in a complex plant vacuolar system. Of the storage compartments, protein storage vacuoles have been best studied because proteins are relatively easy to track in a cell.In many plant cells, separate protein storage and lytic vacuoles coexist. Proteins are delivered to each from the Golgi complex by separate vesicular trafficking pathways, clathrin coated vesicles for the lytic pathway, and dense vesicles (or their equivalents)for the protein storage vacuole pathway. However,in developing seeds and certain other cells, the two pathways converge on the same vacuole. In this instance, for example in protein storage vacuoles in seeds, the resultant organelle is a multivesicular body, where the storage products are partitioned in the "soup " and lytic functions are partitioned into the internal vesicles. Thus the seed protein storage vacuole is a compound organelle,where two functionally distinct compartments exist within the limiting membrane. How proteins are delivered by the two separate vesicular pathways to the two compartments within the organelle is an important unsolved question in cell biology. This project focuses on mechanisms by which proteins are sorted into the protein storage vacuole pathway in the Golgi complex. Results from recent ligand binding experiments indicate that the lumenal domain of a plant RMR protein specifically interacts with the targeting determinants that sort proteins into the protein storage vacuole pathway. RMR proteins are integral membrane proteins that traffic from Golgi to the protein storage vacuole where they are incorporated into a membrane-containing crystalloid within the storage compartment.Thus it is likely they serve as a unique type of sorting receptor.RMR proteins are also expressed in avian and mammalian cells. The association and dissociation constants, and stoichiometry, for binding of RMR protein lumenal domains for a model ligand will be determined. The function of RMR proteins in plants will be assessed by generating antisense knockouts in tobacco and by identifying transposon/T DNA insertions in individual RMR protein genes in Arabidopsis. Motifs in the RMR proteins' cytoplasmic tails responsible for traffic from Golgi to the storage compartment will be identified.A separate experimental strategy will address mechanisms by which the storage compartment crystalloid is formed. Dr. Rogers' laboratory has purified protein storage vacuole crystalloids away from other membranes in the vacuole. Using a proteomics approach, the integral membrane proteins specifically incorporated into PSV crystalloids, and then tonoplast and globoids in B. napus seeds will be identified. The mechanisms by which a tomato storage protein related to 11S globulins that appears to have transmembrane helices is incorporated into crystalloid membranes will be defined.Although protein storage compartments were thought initially to be unique to plant cells, evidence now emerging indicates that animal cells also have a dense vesicle pathway, and that multivesicular body endosomes in animal cells may partition two separate functions within the same organelle. Thus,an understanding of fundamental processes of compartmentation in plant cells may have broader impact in cell biology. The ability to visualize the two compartments easily in protein storage vacuoles and to track proteins in each of the vesicular pathways provides great advantages for use of a plant system in these studies.

植物细胞在液泡中存储各种分子。存储是植物对人类如此重要的重要原因。针对可卡因和海洛因的战争是由于储存液泡的收获而造成的，早晨咖啡被酿造出来释放储存在液泡中的产品，我们花园中的花朵在那里，因为含有液泡中的颜料，家畜的营养和人类的营养最终依赖于植物液泡中存储的蛋白质。植物生物技术中的许多分子都存储在液泡中。只有了解不同的隔室，它们的内部内容和环境，以及如何将蛋白质和膜引导到每个区域，我们才能对单元进行编程以制造和积累所需的产品。将类似于酵母液泡或哺乳动物溶酶体类似的消化室与储存室分开的需要，导致了一个复杂的植物液泡系统。在存储室中，最好研究蛋白质储存液泡，因为蛋白质在细胞中相对易于跟踪。在许多植物细胞中，分开的蛋白质储存和裂解液泡共存。蛋白质通过单独的囊泡运输途径，裂解途径的网格蛋白涂层囊泡以及蛋白质储存液泡途径的密集囊泡（或它们的等效物）从高尔基体配合物中传递到每种蛋白质。但是，在开发种子和某些其他细胞中，这两种途径在同一液泡上汇聚。在这种情况下，例如，在种子中的蛋白质存储液泡中，所得的细胞器是一个多囊体，其中存储产物在“汤”中分配，裂解功能被分配到内部囊泡中。因此，种子蛋白储存液泡是一种化合物细胞器，其中有两个在功能上不同的隔室中存在于极限膜中。在细胞器中，两种单独的囊泡途径如何传递到蛋白质是细胞生物学中的一个重要的未解决问题。该项目的重点是将蛋白质分类为高尔基体综合体中蛋白质储存液途径的机制。最近的配体结合实验的结果表明，植物RMR蛋白的腔内结构域特异性与靶向决定因素相互作用，该靶标确定因素将蛋白质分类为蛋白质储存液泡途径。 RMR蛋白是不可或缺的膜蛋白，从高尔基体到蛋白储存液，将它们掺入储存室内的含膜结晶中。将确定用于模型配体的RMR蛋白腔内结构域结合的缔合和解离常数以及化学计量。 RMR蛋白在植物中的功能将通过在烟草中产生反义敲除，并通过鉴定拟南芥中各个RMR蛋白基因中的转盆/T DNA插入来评估。将确定RMR蛋白质的细胞质尾巴中的基序，以识别负责从高尔基体到储存室的交通。罗杰斯博士的实验室已将蛋白质储存液泡晶体纯化，远离液泡中的其他膜。使用蛋白质组学方法，将鉴定出特异性地掺入PSV晶体中的积分膜蛋白，然后将块状体和球形芽孢杆菌中的球形蛋白识别。将定义与11S球蛋白相关的番茄储存蛋白与11S球蛋白相关的机制，这些机制将被纳入结晶膜中。尽管认为蛋白质储存室最初被认为是植物细胞所独有的，但现在有证据表明，现已出现的证据表明，动物细胞也表明具有多浓密的囊泡小细胞，并且在多个动物的身体中可以分离出两种分离的动物体内，并且可以分离两者。因此，了解植物细胞中隔室基本过程的理解可能对细胞生物学产生更大的影响。在蛋白质储存液泡中轻松可视化这两个隔室的能力以及在每种囊泡途径中跟踪蛋白质的能力，为在这些研究中使用植物系统提供了极大的优势。