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Regulation of Arabidopsis life cycle by the hsp 70 system of the endoplasmic reticulum

内质网hsp 70系统对拟南芥生命周期的调节

基本信息

批准号：
15570034
负责人：
NISHIKAWA Shuh-ichi
金额：
$ 2.37万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570034/
关键词：
endoplasmic reticulum quality control molecular chaperone hsp 70 Arabidopsis thaliana pollen 小胞体 DnaJホモログ BiP

项目摘要

The endoplasmic reticulum(ER) is the entrance of the secretory pathway in eukaryotic cells. Secretory and membrane proteins undergo folding and various modification processes before they are exported to the Golgi apparatus. Quality control mechanisms in the ER monitor these processes to ensure that defective proteins that failed to acquire correct functional structures are not deployed throughout the cells and play important roles in the maintenance of cell homeostasis. The aim of our research is to elucidate functions and mechanisms of the ER quality control system in plant development by analyzing functions of molecular chaperones in the ER, molecular machineries that control ER quality control. BiP, a Hsp 70 family molecular chaperone in the ER, plays a central role both in protein translocation across the ER membrane and ER quality control. In this study, we have developed a system to analyze BiP functions in plant cells using Arabidopsis thaliana.In order to compromise BiP functions in plant cells, we introduced a series of dominant negative mutations into the Arabidopsis BIP gene. When expressed one of the dominant negative mutants in the Arabidopsis culture cells using the 35S promoter, we observed change of subcellular distribution of 12S globulin-GFP fusion protein which can be attributed to its aggregation. We also constructed transgenic plants which express BiP mutants in pollen under the LAT52 promoter. Reciprocal cross experiments suggested that expression of BiP mutants caused pollen sterility. Analyses of yeast mutants identified three J-domain containing protein in the ER as partners for BiP during its function. We identified Arabidopsis homologues of yeast J-domain containing proteins in the ER and constructed their T-DNA insertion mutants.

内质网是真核细胞分泌途径的入口。分泌和膜蛋白在被输出到高尔基体之前经历折叠和各种修饰过程。内质网中的质量控制机制监控这些过程，以确保未能获得正确功能结构的缺陷蛋白不会部署在细胞内，并在维持细胞内稳中发挥重要作用。本研究的目的是通过分析分子伴侣在内质网中的功能，阐明内质网质量控制系统在植物发育中的功能和机制。BIP是内质网中的HSP70家族分子伴侣，在蛋白质跨内质网转运和内质网质量控制中发挥着重要作用。本研究以拟南芥为材料，建立了一套植物细胞Bip功能的分析系统，将一系列显性的负性突变引入拟南芥BIP基因中，以降低Bip在植物细胞中的功能。当用35S启动子在拟南芥培养细胞中表达一个显性负突变体时，我们观察到12S球蛋白-GFP融合蛋白的亚细胞分布发生了变化，这可以归因于它的聚集。我们还构建了在LAT52启动子下在花粉中表达Bip突变体的转基因植株。正反交试验表明，Bip突变体的表达导致了花粉不育。对酵母突变体的分析表明，内质网中的三个含有J结构域的蛋白是Bip功能过程中的合作伙伴。我们鉴定了内质网中含有酵母J结构域蛋白的拟南芥同源物，并构建了它们的T-DNA插入突变体。