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，大约有。700个氨基酸的头部区域和超过1000个氨基酸的长盘绕盘尾。近年来的研究表明，Uso1p参与了er -芽泡在高尔基膜上的分泌途径。从丝状真菌细粒曲霉基因组DNA中分离到USO1同源基因（usoA）。usoA基因编码一个1103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家族由球形头域和长盘绕尾组成的结构特征。我们将染色体usoA基因启动子与诱导型alcA启动子交换，构建了usoA基因条件表达菌株。alcA-usoA突变体在aicA启动子抑制培养基上没有生长。在usoA抑制条件下，突变体停止生长并积累了空泡型羧肽酶CypA的中间形式。这表明UsoAp对生长至关重要，并参与细胞内蛋白质运输。我还观察到aicA-usoA突变体的萌发表现出一系列细胞极性的异常表型，包括出芽模式异常、菌丝膨胀、核聚集和隔。在usoa抑制菌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几丁质的异常定位和取向。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的功能分析，成功构建了一个新的指示细胞内蛋白质转运的细胞极性研究系统。

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.