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Localization mechanisms of membrane proteins in the plant secretory pathway

膜蛋白在植物分泌途径中的定位机制

基本信息

批准号：
15380232
负责人：
MATSUOKA Ken
金额：
$ 10.11万
依托单位：
RIKEN
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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.

为了充分利用植物中较高的聚糖合成能力，利用植物细胞培养或植物培养根生产有价值的生物活性聚糖，修饰的聚糖合成应该定位于高尔基体，因为大多数细胞壁非纤维素聚糖都是在高尔基体合成的。植物也有利用膜锚定氧化还原酶催化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，开发高产量合成这种膜蛋白的方法是必要的。此外，在非适宜环境中，大多数阻碍植物生长的逆境可以通过调节各种转运蛋白来克服。因此，需要开发系统来工程化包括转运蛋白在内的整合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还不如可溶性蛋白，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的cDNA，并分析了它们的定位。结果，获得了以下结果。1)脯氨酰羟化酶是一种定位于高尔基体的II型膜蛋白。高尔基体定位或从内质网的有效输出需要细胞质尾区的碱性残基。2)细胞色素b5和红色荧光蛋白的融合蛋白在细胞中形成稳定的蛋白质聚集体。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的cDNA，并对其中一些进行了定位。