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依赖于内质网/高尔基体的经典分泌途径是胞外分泌的主要途径。传统上认为,经典分泌途径所分泌的蛋白,通常都具有信号肽和高尔基体定位信息。经典分泌途径分泌蛋白由内质网向高尔基体运输,需要COPI小泡作为载体,而布雷非德菌素A(BFA)可以抑制COPI被膜小泡的形成,最终阻断分泌蛋白由内质网向高尔基体的运输。为建立一个简便的鉴别经典分泌途径分泌蛋白的方法,本研究从板栗疫病菌(Cryphonectria parasitica)分泌蛋白组数据库中选择一个不具有信号肽、跨膜区和高尔基体定位信息的氧化还原酶(POR),通过构建POR-GFP融合蛋白,观察其在野生型菌株和在受BFA处理菌株中的细胞定位,发现该融合蛋白的细胞内分布在野生型菌株呈弥散型分布,而在BFA处理菌株时呈聚集型分布,表明其在细胞内由内质网到高尔基体的运输受到了抑制。该结果表明,POR的分泌依赖于经典分泌途径,属于经典途径分泌蛋白。因此,GFP融合蛋白结合BFA处理可以准确鉴定经典途径分泌蛋白。
The classical secretory pathway that relies on the endoplasmic reticulum / Golgi apparatus is the major pathway for extracellular secretion. Traditionally, the proteins secreted by the classical secretory pathway usually have signal peptide and Golgi localization information. Classical secretory pathway secreted proteins are transported from the endoplasmic reticulum to the Golgi and require COPI vesicles as a carrier, whereas bfereotide A (BFA) inhibits the formation of COPI-enveloped vesicles and eventually blocks the secretion of secreted proteins from the endoplasmic reticulum Golgi transport. In order to establish a simple and convenient method to identify secreted proteins from classical secretory pathway, we selected a redox enzyme (POR) without signal peptide, transmembrane domain and Golgi localization information from Cryphonectria parasitica secreted protein database ). By locating the POR-GFP fusion protein and observing its localization in the wild-type strain and in the cells treated with BFA, it was found that the intracellular distribution of the fusion protein was diffusely distributed in the wild-type strain, whereas in the case of the BFA-treated strain Showed an aggregated distribution, indicating that intracellular transport from the endoplasmic reticulum to the Golgi was inhibited. This result indicates that the secretion of POR depends on the classical secretory pathway and belongs to the classical pathway secreted protein. Therefore, GFP fusion protein binding to BFA treatment can accurately identify classical pathway secreted proteins.