板栗疫病菌中一个依赖于经典分泌途径的无信号肽分泌蛋白的鉴定

Identification of a Conventional Secretion Route-dependent Secretory Protein without a Signal Peptide

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 depending on the endoplasmic reticulum/Golgi is the main way for cell exocytosis. Generally, proteins secreted from the classical secretory pathway contain the localization information of the signal peptide and the Golgi. Secretory proteins from the classical pathway transporting from the endoplasmic reticulum to the Golgi utilize the COPI vesicles as the carrier. However, the Brefeldin A （BFA） can inhibit the formation of COPI vesicles to block the transportation of secretory proteins from endoplasmic reticulum to the Golgi. To establish a simple identification method for secreted proteins of the classical secretory pathway, in this study, we chosed an oxidoreductase （POR） without signal peptide, transmembrane region and Golgi localization information from C. parasitica secreted proteome database as the object, by means of constructing POR-GFP fusion protein, observing the cellular localization of the fusion protein in wild-type strains and in the BFA treated strains. The results showed that the fusion protein distributed dispersedly in the wild-type strains, but as aggregated distribution in the BFA treated strains, which indicated that the transportation from the endoplasmic reticulum to the Golgi was inhibited. The results illustrated that the POR was a classical secreted protein and its secretion relied on the classical secretory pathway. Therefore, GFP fusion protein with BFA treated could accurately identify the secreted proteins in the classical secretory pathway.