Mechanisms and Physiological Roles of Endoplacmic Reticulum Quality Control Regulated by Protein Glycosylation

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有缺陷的蛋白质不会部署在整个细胞中。保留在内质网中的错误折叠的蛋白质通过一种称为内质网相关降解(ERAD)的机制降解。本研究的目的是利用发芽酵母和拟南芥对蛋白质进行碳水化合物修饰，以阐明内质网质量控制系统的机制。YEast Mnllp是内质网中的甘露糖苷酶家族蛋白，被认为在错误折叠糖蛋白的ERAD中起底物识别的作用。我们发现，Mnllp通过二硫键与蛋白质二硫键异构酶(PDI)形成复合体。利用Mnllp…突变体进行分析More和PDI表明，Mn11 p-PDI相互作用在ERAD中起重要作用。为了分析ERAD底物识别中蛋白质折叠和碳水化合物修饰之间的关系，我们利用酵母中广泛使用的ERAD底物CPY*开发了新的ERAD底物。使用新开发的ERAD底物的分析表明，CPY*的ERAD所需的N-连接碳水化合物链只有在错误折叠区域时才被识别为ERAD信号。使用哺乳动物和酵母细胞的分析表明，钙粘蛋白/钙网蛋白、EDEM(酵母中的Mnllp)和OS9在碳水化合物链介导的ER质量控制中发挥作用。拟南芥拥有这些内质网质量控制机制的同源基因。我们构建了这些质量控制机制的拟南芥基因敲除突变体，发现AtCNX1和AtCRT1的同时缺失会导致生长迟缓。我们还构建了植物细胞中一种新的ERAD底物AtCPY*-GFP。与酵母CPY*相比，我们发现AtCPY*GFP的ERAD不需要更少的N-连接碳水化合物修饰

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. Misfolded proteins retained in the ER are degraded by a mechanism called endoplasmic reticulum-associated degradation (ERAD) . The aim of our research is to elucidate mechanisms of the ER quality control system mediated by carbohydrate modification of proteins using budding yeast and Arabidopsis thaliana.Yeast Mnllp is a mannosidase family protein in the ER and is proposed to function in the substrate recognition in the ERAD of misfolded glycoproteins. We found that Mnllp forms a complex with protein disulfide isomerase (PDI) through disulfide bonds. Analyses using mutants of Mnllp … More and PDI showed that the Mnll p-PDI interaction is important for ERAD. To analyze relationships between protein folding and carbohydrate modification in substrate recognition in ERAD, we developed new ERAD substrates using CPY*, a widely used ERAD substrate in yeast. Analyses using the newly developed ERAD substrates showed that a N-linked carbohydrate chain required for ERAD of CPY* is recognized as a ERAD signal only when it was in the misfolded domain.Analyses using mammalian and yeast cells showed that calnexin/calreticulin, EDEM (Mnllp in yeast) and OS9 function in carbohydrate chain-mediated ER quality control. Arabidopsis has orthologs of these ER quality control machineries. We constructed Arabidopsis knockout mutants of these quality control machineries and found that simultaneous deletion of AtCNX1 and AtCRT1 resulted in growth retardation. We also constructed AtCPY*-GFP, a novel ERAD substrate in plant cells. In contrast to yeast CPY*, we showed that ERAD of AtCPY*GFP does not require its N-linked carbohydrate modification Less