Control of Storage Protein Biosynthesis by mRNA Targeting

植物种子的主要代谢活动是合成碳（以脂类和淀粉的形式）和氮（以贮藏蛋白的形式）储备。 尽管负责这些大分子生物合成的生化过程似乎是独立的过程，但多年来收集的证据表明并非如此。 碳和氮代谢是相互关联的，如在这两个过程之一中基因表达改变的确定的遗传突变体和转基因植物所表现出的几乎平行的反应所证明的。 负责碳和氮储备的这种协调表达的机制尚不清楚。 本基金拟开展一系列研究，研究氮代谢的一个方面，即水稻胚乳发育过程中贮藏蛋白生物合成的控制。 水稻是研究贮藏蛋白生物合成（及其与淀粉合成的关系）的极好系统，因为它是合成和积累两种主要类别的贮藏蛋白（醇溶谷蛋白和谷蛋白，后者是11 S球蛋白）的少数植物之一。 此外，大米将这些蛋白质储存在不同的亚细胞区室中。 醇溶谷蛋白直接沉积在内质网（ER）腔内的脑池内的蛋白质颗粒，而谷蛋白通过ER运输并包装在蛋白质储存泡中。 本实验室先前的研究已经证明，编码这两类储存蛋白的RNA并不以随机的方式定位在ER上。 相反，醇溶谷蛋白mRNA高度富集在结合醇溶谷蛋白蛋白体（PB）的ER膜上，而谷蛋白mRNA主要分布在池状ER上。 大多数醇溶谷蛋白PB分布在皮层的质膜附近，这是一个富含细胞骨架的区域。 醇溶谷蛋白mRNA定位于皮层区域，距离细胞核7-15 μ m，表明醇溶谷蛋白RNA本身而不是编码的蛋白质靶向醇溶谷蛋白PB。 事实上，正在进行的研究结果在转基因水稻中的合成醇溶谷蛋白RNA转录本的定位表明，这些RNA的目标是醇溶谷蛋白PB，因为它们含有特定的“邮政编码”信号。 为了更好地理解该过程，描述了尝试鉴定这些“邮政编码”的实验，并鉴定和表征邮政编码结合蛋白，其负责醇溶谷蛋白RNA的运输和锚定到醇溶谷蛋白PB。 还将努力重建醇溶蛋白RNA运动在培养和发展胚乳细胞，这将提供细胞内的高速公路RNA运动的细胞细节，并提供线索的性质的分子马达负责RNA运动。 该项目的第二个总体目标是确定谷蛋白RNA是否也具有将其靶向不同ER亚域的“邮政编码”，并确定细胞中醇溶谷蛋白和谷蛋白RNA定位之间的空间关系。 未来的研究将致力于编码淀粉生物合成酶的RNA的细胞内定位。 将采取综合方法来实现这些目标，并将结合使用生物化学，细胞，分子和超微结构技术。 这项研究将提供关于RNA和蛋白质如何在植物细胞中分类以及ER如何分化为具有特定功能的独特结构域的新信息。 此外，它将导致对可能控制碳和氮的利用及其转化为储存储备的机制的新见解。

The major metabolic activities of plant seeds are directed toward the synthesis of carbon (in the form of lipids and starch) and nitrogen (in the form of storage proteins) reserves. Although the biochemical processes responsible for the biosynthesis of these macromolecules appear to be independent processes, evidence gathered over the years have indicated otherwise. Carbon and nitrogen metabolism are inter-related as evidenced by the almost parallel responses exhibited by defined genetic mutants and transgenic plants altered in gene expression in one of these two processes. The mechanisms responsible for this coordinated expression of carbon and nitrogen reserves are not known. This grant proposes a series of studies to study one aspect of nitrogen metabolism, i.e. the control of storage protein biosynthesis in developing rice endosperm. Rice is an excellent system to study storage protein biosynthesis (and its relationship to starch synthesis) in that it is one of the few plants that synthesize and accumulate both major classes of storage proteins, prolamines and glutelins, the latter an 11S globulin. Moreover, rice stores these proteins in different subcellular compartments. Prolamines are deposited directly as protein intracisternal granules within the endoplasmic reticulum (ER) lumen, whereas glutelins are transported through the ER and packaged in a protein storage vacuole. Previous studies from this laboratory have demonstrated that the RNAs encoding these two classes of storage proteins are not localized in a stochastic fashion on the ER. Instead, prolamine mRNAs are highly enriched on the ER membranes that bound the prolamine protein bodies (PBs) whereas glutelin mRNAs predominant on the cisternal ER. The majority of the prolamine PBs is distributed near the plasma membrane in the cortex, a region enriched in cytoskeleton. The localization of prolamine mRNAs to the cortical region, a distance of 7-15 um from the nucleus, suggests that the prolamine RNA itself, and not the coded protein, is targeted to the prolamine PBs. Indeed, results from ongoing studies on the localization of synthetic prolamine RNA transcripts in transgenic rice indicate that these RNAs are targeted to the prolamine PBs because they contain specific "zip code" signals. To better understand this process, experiments are described that will attempt to identify these "zip codes", and to identify and characterize the zip code binding proteins, which are responsible for the transport and anchoring of the prolamine RNAs to the prolamine PBs. Efforts will also be made to reconstruct prolamine RNA movement in cultured and developing endosperm cells, which will provide cellular details on the intracellular highways for RNA movement and provide clues on the nature of the molecular motor responsible for RNA movement. A second general goal of the project is to determine whether glutelin RNAs also possess "zip codes" that target it to a different ER subdomain and to determine the spatial relationship between prolamine and glutelin RNA localization in the cell. Future studies will address the intracellular location of RNAs that code for enzymes involved in starch biosynthesis. An integrated approach will be taken to achieve these goals, and will incorporate the use of biochemical, cellular, molecular and ultrastructural techniques. This study will provide new information on how RNAs and, in turn, proteins are sorted in plant cells and how the ER can differentiate into unique domains with specific functions. Moreover, it will lead to new insights into the mechanisms that may control utilization of carbon and nitrogen and their conversion into storage reserves.