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

点击翻译按钮获取中文摘要

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.