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Analysis of the mechanisms of protein sorting and protein modification in the secretory pathway of higher plants

高等植物分泌途径中蛋白质分选和蛋白质修饰机制分析

基本信息

批准号：
13460152
负责人：
MATSUOKA Ken
金额：
$ 9.92万
依托单位：
RIKEN (The Institute of Physical and Chemical Research)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Genetically modified crops are expected as factories to produce useful proteins of animal origin because of the lower cost of production than animals and microorganisms. Most of the useful proteins from animals are secretory proteins and these proteins are modified with glycans in the Golgi appratus after synthesis at the ER, and then trasnported to the vacuole/lysosome or extracellular space. Molecular mechanism of protein sorting and protein modification in the Golgi apparatus are different in organisms belonging to different kingdom ; thus, production of these animal-oriented proteins in plants as active forms require the control of plant secretory pathway by suppressiing the plant sorting and/or modification mechanism. In this research, we conducted the analysis of protein sorting apparatus from yeasts as well as the analysis of protein sorting in plants by developing in vitro system to generate transport vesicles from isolated membranes and cytosol. We also carried out analyses on glycosylation and proline hydroxylation of secretory proteins in plants. For these purposes we aimed to develop a novel methods to analyze membrane proteins. Based on these works we got the following results : 1) Definition of the topological orientation of coat proteins of COPII vesicles. 2) Role of a GTP-ase Arf1p on the protein transport in plants. 3) Characterization of proryl hydroxylase from plants. In addition we developed a rapid system to determine the solubilization condition of membrane proteins.

转基因作物有望成为生产有用的动物来源蛋白质的工厂，因为其生产成本低于动物和微生物。大多数来自动物的有用蛋白质是分泌蛋白，并且这些蛋白质在内质网合成后在高尔基体中被聚糖修饰，然后转运到液泡/溶酶体或细胞外空间。不同界的生物体在高尔基体中的蛋白质分选和蛋白质修饰的分子机制是不同的;因此，在植物中以活性形式产生这些动物导向蛋白质需要通过抑制植物分选和/或修饰机制来控制植物分泌途径。在本研究中，我们进行了蛋白质分选装置从酵母的分析，以及蛋白质分选在植物中的分析，通过开发在体外系统，以产生运输囊泡从分离的膜和胞质溶胶。我们还对植物分泌蛋白的糖基化和脯氨酸羟基化进行了分析。为此，我们的目标是发展一种新的方法来分析膜蛋白。在此基础上，我们得到了以下结果：1）确定了COPII囊泡外壳蛋白的拓扑取向。2)GTP酶Arf 1 p在植物蛋白质转运中的作用3)植物脯氨酰羟化酶的性质研究。此外，我们还建立了一个快速测定膜蛋白溶解条件的系统。