Targeting of Vacuolar Proteins In Plant Cells

这项工作的主要目的是鉴定使液泡蛋白能够靶向植物细胞液泡的蛋白质结构域，并鉴定它们与之相互作用的受体。这项工作将集中于存在于豆类（Phaseolus vulgaris）种子中的三种液泡蛋白、植物血凝素、phaseolin和cx-淀粉酶抑制剂，并将使用两种不同的转基因系统，酵母酿酒酵母和烟草（Nicotiana tabacum）。此外，我们将尝试用农杆菌转化豆类，以便研究向液泡的转运。这项工作将利用这三种蛋白的部分与酵母转化酶基因（一种分泌蛋白）之间的翻译融合来描绘靶向结构域。诱变将与基于计算机的序列比较一起使用，以确定关键残基。受体的鉴定将依赖于使用这些蛋白质和溶解膜组分的靶向结构域的亲和研究（无论是柱或凝胶/印迹结合程序）。一旦受体被分离出来，我们将尝试克隆该基因，并进行免疫细胞化学工作来研究其亚细胞分布。该项目将为我们理解细胞蛋白如何到达细胞内特定功能位点提供重要的基础信息。它还将提供有关种子储存蛋白如何到达细胞内储存位置的重要信息，这在营养“改良”种子和谷物的基因工程方面具有实用价值。

The major objective of this work is to identify the protein domain that allows vacuolar proteins to be targeted to the vacuoles of plant cells, and to identify the receptor(s) with which they interact. The work will concentrate on three vacuolar proteins present in bean (Phaseolus vulgaris) seeds, phytohemagglutinin, phaseolin, and cx-amylase inhibitor and will use two different transgenic systems, the yeast Saccharomyces cerevisiae and tobacco (Nicotiana tabacum). In addition, we will attempt to transform bean with Agrobacterium tumefaciens to be in a position to study transport to the vacuole. The work will utilize translational fusions between portions of these three proteins and the yeast invertase gene (a secreted protein) to delineate the targeting domain. Mutagenesis will be used together with computer-based sequence comparisons to identify the critical residues. Identification of the receptor will rely on affinity studies (either columns or gel/blot binding procedures) using the targeting domains of these proteins and solubilized membrane fractions. Once the receptor has been isolated, we will attempt to clone the gene and carry out immunocytochemical work to study its subcellular distribution. This project will provide important basic information relevant to our understanding of how cellular proteins get to their specific functional sites within the cell. It will also provide important information concerning how seed storage proteins get to the sites of storage within the cell, which is of practical value in terms of genetic engineering of nutritionally "improved" seeds and grains.