Localization mechanisms of membrane proteins in the plant secretory pathway

为了利用植物中较高的多糖合成能力，利用植物细胞培养或植物培养根生产有价值的、具有生物活性的多糖，改性多糖的合成应局限于高尔基体，大部分细胞壁非纤维素聚糖在高尔基体中合成。植物也有利用膜锚定氧化还原酶分解复杂化合物的代谢途径。这些酶的工业应用需要高水平的蛋白质生产。因此，开发高质量合成膜蛋白的方法是必要的。此外，在非最佳环境中，大多数阻碍植物生长的逆境都可以通过各种转运体的调节来克服。因此，需要开发系统来设计包括转运蛋白在内的整体膜蛋白的定位和稳定性。然而，目前对膜蛋白定位机制的了解比对可溶性蛋白定位机制的了解还少。因此，本研究以细胞色素b5和脯氨酰羟化酶为主要工具，旨在阐明植物细胞中膜蛋白定位和降解的信号和机制。除此之外，我们还从烟草中克隆了几个膜转运蛋白cdna并分析了它们的定位。结果得到如下结果：1)脯氨酸羟化酶是一种定位于高尔基体的ii型膜蛋白。内质网的高尔基定位或有效输出需要细胞质尾部的基本残基。2)细胞色素b5的融合蛋白与一个红色荧光蛋白在细胞内形成稳定的蛋白聚集体。聚集体的形成取决于RFP的四聚体性质。3)从烟草BY-2细胞中克隆了几种转运蛋白和其他膜蛋白cdna，并对其中一些进行了定位鉴定。

In order to utilize higher ability of the glycan synthesis in plants and to produce valuable and bioactive glycans using plant cell culture or plant cultured roots, modified glycan syntheses should be localized to the Golgi apparatus, where most of the cell wall non-cellulose glycans synthesized. Plants also have metabolic pathways for the catabolism of complex compounds using membrane-anchored oxidoreductases. The industrial use of these enzymes requires high-level production of the proteins. Thus developing procedures to synthesize such membrane proteins with high quantity is necessary. Moreover, most of the stresses that prevent the growth of plants in non-optimum environment can overcome by the modulation of various transporters. Thus developing systems to engineer localizations and stabilities of integral membrane proteins including transporters are requested. At present, however, knowledge on the localization mechanism of membrane proteins are scarcer than the one of soluble proteins.Therefore, in this work we aimed to clarify the signals and mechanisms of the localization and degradation of membrane proteins in plant cells using cytochrome b5 and prolyl hydroxylase as major tools. In addition to this work we cloned several membrane transporter cDNAs from tobacco and analyzed their localization. As a result, the following results were obtained. 1)Prolylhydroxylase is a typeII membrane protein localizing to the Golgi apparatus. The Golgi localization or efficient export from the ER requires basic residues in the cytoplasmic tail. 2)Fusion protein of cytochrome b5 and a red fluorescent protein form stable protein aggregate in the cell. Formation of the aggregate depends on the tetrameric nature of RFP. 3)From tobacco BY-2 cells several transporter and other membrane protein cDNAs were cloned and localization of some of them are characterized.