Involvement of AAA ATPase AipA in endocytosis of the arginine permease AoCan1 depending on AoAbp1 in Aspergillus oryzae

Abstract AAA (ATPases associated with diverse cellular activities) ATPases widely exist in many organisms and function in various organelles. However, there is little information about AAA ATPase functioning in endocytosis. In the filamentous fungus Aspergillus oryzae, we previously discovered a putative AAA ATPase AipA (AoAbp1 interacting protein) that would be involved in endocytosis. Here, we further examined the function of AipA and AoAbp1 in endocytosis using enhanced green fluorescent protein (EGFP)-tagged arginine permease AoCan1 as an endocytic marker. In the ΔaipA strain, endocytosis of AoCan1-EGFP was more facilitated than the control strain, suggesting that AipA negatively regulates endocytosis. Additionally, the localization of F-actin, visualized by Lifeact-EGFP, was concentrated at the hyphal tip in the ΔaipA strain than the control strain, suggesting that endocytosis was promoted due to enhanced actin polymerization in the ΔaipA strain. In contrast, in the ΔAoabp1 strain, endocytosis of AoCan1-EGFP was delayed compared with the control strain, suggesting that AoAbp1 positively functions in endocytosis. In addition, in the ΔaipAΔAoabp1 strain, endocytosis of AoCan1-EGFP was also delayed. AipA localized at the endocytic collar of the hyphal tip, only in the presence of AoAbp1, suggesting that AipA functions downstream of AoAbp1 in endocytosis. Moreover, we investigated the aipA-overexpressing strain, and found that endocytosis of AoCan1-EGFP was inhibited. Furthermore, we examined strains expressing aipAK542A or aipAE596Q, which decreased ATPase activity, in the backgrounds of complementation or overexpression, respectively, and found that AoCan1-EGFP endocytosis was promoted. These results suggested that AAA ATPase activity of AipA is important for its function in endocytosis.