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

转基因作物有望作为工厂生产动物来源的有用蛋白质，因为其生产成本低于动物和微生物。大多数动物有用的蛋白质是分泌性蛋白质，这些蛋白质在内质网合成后被高尔基体中的葡聚糖修饰，然后运输到液泡/溶酶体或细胞外空间。高尔基体中蛋白质分选和修饰的分子机制在不同的生物界中是不同的，因此，在植物中生产这些面向动物的蛋白质作为活性形式，需要通过抑制植物的分选和/或修饰机制来控制植物的分泌途径。在本研究中，我们对酵母菌的蛋白质分选装置进行了分析，并通过开发从分离的膜和胞浆中产生运输囊泡的体外系统来分析植物的蛋白质分选。我们还对植物分泌蛋白的糖基化和脯氨酸羟化进行了分析。为此，我们的目标是开发一种分析膜蛋白的新方法。在这些工作的基础上，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Arf1p在植物蛋白质运输中的作用。3)植物丙基羟基酶的性质。此外，我们还开发了一种快速测定膜蛋白增溶条件的系统。

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.