Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae

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.The filamentous fungus Aspergillus oryzae has been used in industrial fermentation processes and is regarded to be safe for humans. A. oryzae can secrete several proteins, such as alpha-amylase, into the medium. Thus, A. oryzae is a potential host for heterologous protein production. Since the completion of A. oryzae genome sequencing (18) in recent years, many applied and basic studies have been conducted on A. oryzae using its genome sequencing data. In particular, studies on vesicular trafficking, including the secretory pathway, are of increasing importance because they are closely related to protein production. For example, endoplasmic reticulum and vacuole dynamics and systematic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein analyses have been performed in A. oryzae (16, 19, 23, 30, 31, 32). However, endocytosis, an intracellular trafficking pathway, has not been studied as well in A. oryzae as in other filamentous fungi.Endocytosis is an important cellular process that occurs, for example, in signal transduction and reconstruction of cell polarity and is conserved in eukaryotic cells. The detailed mechanism of endocytosis has been well studied in model organisms such as yeasts. Many proteins are involved in the endocytic process, which is regulated spatiotemporally (12). Saccharomyces cerevisiae END4/SLA2 (synthetic lethal with ABP1) is an endocytosis-associated gene that has been studied in detail (3, 6, 22, 27, 35, 43, 44). End4p/Sla2p is essential for fluid-phase and receptor-mediated endocytosis and actin assembly and polarization (27). The protein has the epsin N-terminal homology (ENTH) and the AP180 N-terminal homology (ANTH) domains, which bind to phosphatidylinositol-4,5-bisphosphate in the plasma membrane in the N-terminal region, and the I/LWEQ domain, which is proposed to be the actin-binding domain in the C-terminal region; it also functions as an adaptor that connects the invaginated plasma membrane and actin cytoskeleton, which plays an important role in endocytosis, to generate force for invaginating the plasma membrane into the intracellular space and forming endocytic pits (13, 33). Abp1p (actin-binding protein) forms actin patches by polymerization of the actin cytoskeleton. It is suggested that endocytosis occurs at the sites in which Abp1p localizes, i.e., cortical actin patches (21, 22). Hence, Abp1p has been used as a tool to investigate the subcellular space in which endocytosis occurs (21).Establishing the existence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis (28). However, it has been confirmed 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 uric acid-xanthine permease]) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis (8, 25). Moreover, recently, in Aspergillus nidulans, the localization of components required for endocytosis has been analyzed in living hyphae (1, 37, 41). ActA and FimA, which are actin and fimbrin, respectively, are mostly localized in the hyphal tip region (41). Furthermore, AbpA, an actin-binding protein, is primarily localized in the apical region and is used as an endocytic site marker. AmpA, the amphiphysin homolog in A. nidulans, and SlaB, the End4p/Sla2p homolog, are also localized in sites in which AbpA is localized (1). These endocytic components are localized near the hyphal tip regions but slightly away from the apex where exocytosis preferentially occurs (37). Although the occurrence of endocytosis was clearly demonstrated and the localization of endocytic components was analyzed, the physiological importance of endocytosis in filamentous fungi still remains largely unaddressed.In this report, we analyzed the physiological significance of endocytosis by generating strains that conditionally express A. oryzae end4 (Aoend4), the A. oryzae homolog of S. cerevisiae END4/SLA2. Hyphae grown in the Aoend4-repressed condition displayed aberrant morphology; endocytic defects in AoUapC-EGFP and FM4-64; abnormal apical recycling of EGFP-fused AoSnc1, which is a vesicle SNARE required for secretion; and abnormal cell wall synthesis. These results suggest that endocytosis plays crucial roles in the physiology of hyphal growth.