Plant Vacuole Biogenesis and Function

植物细胞在液泡中储存各种各样的分子。储存是植物对人类如此重要的核心原因。与可卡因和海洛因的战争源于储存液泡中产品的收获，早晨煮咖啡是为了释放储存在液泡中的产品，我们花园里的鲜花是因为储存在液泡中的色素而存在的，家畜和人类的营养最终依赖于储存在植物液泡中的蛋白质。植物生物技术中的许多分子都储存在液泡中。只有了解了不同的隔间，它们的内部内容物和环境，以及蛋白质和膜是如何到达每个隔间的，我们才能对细胞进行编程，以制造和积累所需的产品。从储藏室中分离出类似于酵母液泡或哺乳动物溶酶体的消化室的需要导致了一个复杂的植物液泡系统。在储藏室中，蛋白质储存液泡是研究得最好的，因为蛋白质在细胞中相对容易追踪。在许多植物细胞中，分离的蛋白质储存和裂解的液泡共存。蛋白质从高尔基复合体通过单独的囊泡运输途径传递到每个蛋白质，为裂解途径提供笼罩的囊泡，为蛋白质储存液泡途径提供致密的囊泡(或其等价物)。然而，在发育中的种子和某些其他细胞中，这两条途径会聚在同一个液泡上。在这种情况下，例如，在种子中的蛋白质存储液泡中，所得到的细胞器是多囊泡体，其中存储产物被分割成“汤”，并且裂解功能被分割成内部囊泡。因此，种子蛋白质储存液泡是一个复合细胞器，在限制膜内存在两个功能不同的隔室。蛋白质如何通过两条不同的囊泡途径输送到细胞器内的两个隔室，是细胞生物学中一个重要的悬而未决的问题。本项目主要研究高尔基复合体中蛋白质被分选到蛋白质储存液泡途径中的机制。最近的配基结合实验结果表明，植物RMR蛋白的管腔结构域与靶向决定因素特异地相互作用，这些决定因素将蛋白质分类到蛋白质储存液泡途径中。RMR蛋白是一种完整的膜蛋白，它们从高尔基体运输到蛋白质储存液泡，在那里它们被结合到储存室内的含有膜的晶体中。因此，它们可能是一种独特的分类受体。RMR蛋白也在鸟类和哺乳动物细胞中表达。结合模型配体的RMR蛋白管腔结构域的结合和解离常数以及化学计量比将被确定。RMR蛋白在植物中的功能将通过在烟草中产生反义敲除和通过鉴定在拟南芥中单个RMR蛋白基因中的转座子/T DNA插入来评估。将确定RMR蛋白细胞质尾巴中负责从高尔基体到储存室的运输的基序。另一个单独的实验策略将解决储存室晶体形成的机制。罗杰斯博士的实验室已经从液泡中的其他膜中提纯了蛋白质存储液泡晶体。利用蛋白质组学方法，鉴定出甘蓝型油菜种子中特异整合到PSV晶体中的整合膜蛋白，以及液泡体和球体。与11S球蛋白相关的番茄贮藏蛋白似乎具有跨膜螺旋的机制将被定义。虽然蛋白质储存间隔最初被认为是植物细胞所独有的，但现在的证据表明，动物细胞也有致密的囊泡途径，动物细胞中的多囊体内内体可能在同一细胞器内划分两个独立的功能。因此，了解植物细胞分室的基本过程可能会对细胞生物学产生更广泛的影响。在蛋白质储存液泡中容易地显示两个隔室的能力，以及跟踪每个囊泡通路中的蛋白质的能力，为在这些研究中使用植物系统提供了巨大的优势。

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.