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

由于生产成本低于动物和微生物，转基因作物有望作为工厂生产有用的动物源性蛋白质。动物体内的有用蛋白大多是分泌蛋白，这些蛋白在内质网合成后，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。不同生物的高尔基体中蛋白质分选和蛋白质修饰的分子机制不同；因此，这些面向动物的蛋白在植物体内作为活性形式产生，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，我们对酵母的蛋白质分选装置进行了分析，并通过建立离体系统，从分离的膜和细胞质中产生运输囊泡，对植物的蛋白质分选进行了分析。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，我们旨在开发一种新的膜蛋白分析方法。基于这些工作，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在植物蛋白转运中的作用。3)植物中丙基羟化酶的表征。此外，我们还开发了一套快速测定膜蛋白溶解条件的系统。

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.