Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae
Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae
复制标题
DOI：
10.1128/ec.00207-08
复制
发表时间：
2009-01-01
期刊：
影响因子：
--
通讯作者：
Kitamoto, Katsuhiko
中科院分区：
文献类型：
--
作者：
Higuchi, Yujiro;Shoji, Jun-ya;Kitamoto, Katsuhiko
在过去，确定丝状真菌中内吞作用的发生是难以捉摸的，主要是由于缺乏可靠的内吞作用指标。然而，最近显示荧光染料N-（3-三乙基铵丙基）4-（对二乙基-氨基苯基-己三烯基）吡啶鎓二溴化物（FM 4 -64）和融合到增强型绿色荧光蛋白（EGFP）的质膜蛋白AoUapC（曲霉属AoUapC）通过胞吞作用从质膜内化。虽然内吞作用的发生已被清楚地证明，但其在丝状真菌中的生理重要性仍然在很大程度上未得到解决。我们产生了一个菌株，其中A。Aoend4（Aoend4）、A.酿酒酵母END 4/SLA 2的Aoend 4同源物在可调控的thiA启动子的控制下从Aoend 4基因座表达。该菌株的生长受到严重损害，其菌丝形态在Aoend 4抑制条件下发生改变。此外，在Aoend 4抑制条件下，FM 4 -64和AoUapC-EGFP均未内化，表明内吞作用缺陷。此外，本地化的分泌可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）是异常的Aoend 4抑制条件。通过calcofluor白色染色和透射电子显微镜分析也观察到细胞壁组分的异常积累，并且编码细胞壁构建酶的几个基因被上调，这表明在Aoend 4抑制条件下细胞壁合成的调节是异常的，而Aopil 1破坏物不显示在Aoend 4抑制条件下表现出的表型。我们的研究结果有力地表明，内吞作用是至关重要的菌丝顶端生长的丝状真菌。
Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed. We generated a strain in which A. oryzae end4 (Aoend4), the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2, was expressed from the Aoend4 locus under the control of a regulatable thiA promoter. The growth of this strain was severely impaired, and its hyphal morphology was altered in the Aoend4-repressed condition. Moreover, in the Aoend4-repressed condition, neither FM4-64 nor AoUapC-EGFP was internalized, indicating defective endocytosis. Furthermore, the localization of a secretory soluble N- ethylmaleimide-sensitive factor attachment protein receptor (SNARE) was abnormal in the Aoend4-repressed condition. Aberrant accumulation of cell wall components was also observed by calcofluor white staining and transmission electron microscopy analysis, and several genes that encode cell wall-building enzymes were upregulated, indicating that the regulation of cell wall synthesis is abnormal in the Aoend4-repressed condition, whereas Aopil1 disruptants do not display the phenotype exhibited in the Aoend4-repressed condition. Our results strongly suggest that endocytosis is crucial for the hyphal tip growth in filamentous fungi.