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)。尽管碳和氮代谢看起来是相互独立的过程，但碳和氮代谢是相互关联的，在这两个过程中的一个过程中，定义的基因突变体和基因表达改变的转基因植物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。这项拨款申请提出了一系列关于氮代谢的一个方面的研究，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的优良系统，因为它合成和积累了两类主要的贮藏蛋白，醇溶蛋白和谷蛋白，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，而谷蛋白则通过内膜系统运输到蛋白质储存液泡。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在结合醇溶蛋白小体(PBS)的内质网膜上高度富含，而谷蛋白mRNAs在脑池内质网中占主导地位。醇溶蛋白mRNAs与醇溶蛋白PBS的定位是通过核内RNA的定向转运实现的。为了更好地了解这一过程，将进行研究，以确定指导RNA运输和定位的核苷酸序列，并鉴定和表征负责将醇溶蛋白和谷蛋白RNA分别运输和锚定到醇溶蛋白PBS和脑池内质网的蛋白质。由于RNA运输是通过颗粒的形成和运动发生的，因此将努力分离这些RNA运输颗粒，并确定哪些其他RNA物种可能共运输。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 appearing to be independent processes, carbon and nitrogen metabolism are inter-related as evidenced by the near 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 application proposes a series of studies on 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 as it synthesizes and accumulates 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 endomembrane system to 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 bind the prolamine protein bodies (PBs) whereas glutelin mRNAs are predominant on the cisternal ER. The localization of prolamine mRNAs to the prolamine PBs occurs by directed RNA transport from the nucleus. To better understand this process, studies will be undertaken to identify the nucleotide sequences that direct RNA transport and localization and to identify and characterize proteins, which are responsible for the transport and anchoring of prolamine and glutelin RNAs to the prolamine PBs and cisternal ER, respectively. As RNA transport occurs by the formation and movement of particles, efforts will be made to isolate these RNA transport particles and determine what other RNA species may be co-transported. 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.