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

内质网是真核细胞分泌途径的入口。分泌蛋白和膜蛋白在输出到高尔基体之前经历折叠和各种修饰过程。ER中的质量控制机制监测这些过程，以确保未能获得正确功能结构的缺陷蛋白质不会在整个细胞中部署，并在维持细胞稳态中发挥重要作用。本研究的目的是通过分析内质网中分子伴侣的功能，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Hsp 70家族分子伴侣，在蛋白质跨内质网膜转运和内质网质量控制中起核心作用。在本研究中，我们利用拟南芥（Arabidopsis thaliana）建立了一个分析植物细胞BiP功能的系统，并在拟南芥BIP基因中引入了一系列显性负突变，以降低植物细胞BiP功能。当用35 S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12 S球蛋白-GFP融合蛋白的亚细胞分布的变化，这可以归因于它的聚集。我们还构建了转基因植物，表达BiP突变体的花粉下的LAT 52启动子。正反交试验表明，BiP突变体的表达导致花粉不育。酵母突变体的分析确定了三个J-结构域的蛋白质在ER作为合作伙伴的BiP在其功能。我们确定了拟南芥同源的酵母J结构域蛋白质ER和构建其T-DNA插入突变体。

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.