Control of Storage Protein Biosynthesis by mRNA Targeting

植物种子的主要代谢活动是合成碳(以脂肪和淀粉的形式)和氮(以贮藏蛋白质的形式)。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。碳和氮的代谢是相互关联的，明确的基因突变体和转基因植物在这两个过程中的一个过程中表现出的几乎平行的反应就证明了这一点。这种碳和氮储量协调表达的机制尚不清楚。这项拨款提出了一系列研究，以研究氮代谢的一个方面，即控制水稻胚乳发育过程中贮藏蛋白的生物合成。水稻是研究贮藏蛋白生物合成(及其与淀粉合成的关系)的一个很好的系统，因为它是少数几种合成和积累主要贮藏蛋白醇溶蛋白和谷蛋白的植物之一，后者是一种11S球蛋白。此外，大米将这些蛋白质储存在不同的亚细胞隔间。醇溶蛋白直接以蛋白质颗粒的形式沉积在内质网(ER)管腔内，而谷蛋白则通过内质网运输并包装在蛋白质存储液泡中。本实验室以前的研究已经证明，编码这两类储存蛋白的RNA不是以随机方式定位在内质网上的。相反，醇溶蛋白mRNAs高度富含在结合醇溶蛋白小体(PBS)的内质网膜上，而谷蛋白mRNAs主要分布在脑池内质网上。大多数醇溶蛋白PBS分布在皮质的质膜附近，这是一个细胞骨架丰富的区域。醇溶蛋白mRNAs定位于皮质区，与核的距离为7-15微米，这表明醇溶蛋白RNA本身，而不是编码的蛋白质，针对醇溶蛋白PBS。事实上，正在进行的关于转基因水稻中合成醇溶蛋白RNA转录本定位的研究结果表明，这些RNA针对醇溶蛋白PBS，因为它们包含特定的“邮政编码”信号。为了更好地理解这一过程，描述了一些实验，这些实验将试图识别这些“邮政编码”，并鉴定和表征负责将醇溶蛋白RNA运输和锚定到醇溶蛋白PBS的邮政编码结合蛋白。还将努力重建培养和发育中的胚乳细胞中的醇溶蛋白RNA运动，这将为RNA运动的细胞内高速公路提供细胞细节，并为解释负责RNA运动的分子马达的性质提供线索。该项目的第二个总体目标是确定谷蛋白RNA是否也具有针对不同ER亚域的“邮政编码”，并确定醇溶蛋白和谷蛋白RNA在细胞中定位的空间关系。未来的研究将解决编码参与淀粉生物合成的酶的RNA在细胞内的位置。将采取综合方法来实现这些目标，并将结合使用生化、细胞、分子和超微结构技术。这项研究将提供新的信息，关于RNA和蛋白质在植物细胞中是如何分类的，以及内质网如何分化成具有特定功能的独特区域。此外，它还将为控制碳和氮的利用及其转化为储存储备的机制带来新的见解。

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.