Functional analysis of the usoA gene involved in the protein secretion in Aspergillus nidulans

Uso1p是酵母菌分泌机制的组成部分之一，约有1/3。头部有700个氨基酸，长螺旋尾巴有1000多个氨基酸。最近的研究表明，Uso1p参与了内质网萌发的小泡在分泌途径中与高尔基体膜的连接。我们从丝状真菌nidulans的基因组DNA中分离到了USO1同源基因(UsoA)。UsoA基因编码1,103个氨基酸的蛋白质，与酵母Uso1p和哺乳动物p115有25%-30%的相似性。USOAP保留了Uso1p家族由球形头部结构域和长螺旋尾巴组成的结构特征。以染色体上的usoA基因启动子为诱导型alca启动子，构建了usoA基因条件表达菌株。突变株Alca-usoA在抑制AICA启动子的培养基上不生长。在usoA抑制条件下，突变株停止生长，并积累了中间形态的液泡型羧肽酶CypA。这表明USOAP对细胞的生长是必需的，并且参与了细胞内蛋白质的运输。我还观察到，AicA-usoA突变体的种苗表现出一系列异常的细胞极性表型，包括异常的萌发模式、菌丝膨胀、簇状核和隔膜。在usoA抑制株中，还观察到细胞骨架成分肌动蛋白和微管蛋白以及细胞壁成分甲壳素的异常定位和方向。这些发现表明，USOAP在细胞极性的形成和维持中起作用。我通过对丝状真菌中USOAP的聚焦功能分析，成功地构建了一种新的细胞极性指示细胞内蛋白质转运的研究体系。

Uso1p, one of the components of secretory machinery in yeast, has approx. 700-amino acids of head domain and over 1,000-amino acids long coiled-coil tail. Recent researches are revealing that Uso1p is involved in the tethering of the ER-budded vesicles to the Golgi membrane in the secretory pathway. We have isolated the USO1 homologous gene (usoA) from a genomic DNA of filamentous fungus Aspergillus nidulans. The usoA gene encodes a 1,103 amino acids protein with 25 - 30% similarity to yeast Uso1p and mammalian p115. UsoAp conserves structural feature of Uso1p family composed of globular head domain and long coiled-coil tail. We constructed the usoA gene conditional expressing strain by exchanging the promoter of the chromosomal usoA gene for the inducibie alcA promoter. The alcA-usoA mutant showed no growth on the aicA promoter-repressing medium. Under the usoA repressing condition, the mutant stopped the growth and accumulated the intermediate form of vacuolar carboxypeptidase CypA. It indicates that UsoAp is essential for growth and involves in the intracellular protein transport. I also observed that the germlings of aicA-usoA mutant showed a series of aberrant phenotypes of cellular polarity including abnormal budding pattern, expanding mycelia, clustered nuclei and septa. Abnormal localizations and orientations of cytoskeletal components, actin and tubulin, and a cell wall component, chitin, were also observed in the usoA-repressed strain. These finding suggested that UsoAp plays a role in formation and maintenance of the cellular polarity. I have successfully constructed a novel investigating system for cellular polarity indicating intracellular protein transport by focusing functional analysis of UsoAp in filamentous fungi.