Functional analysis of the putative AAA ATPase AipA localizing at the endocytic sites in the filamentous fungus Aspergillus oryzae

Abstract

We searched for novel components involved in Aspergillus oryzae endocytosis by yeast two-hybrid (YTH) screening. Using the endocytic marker protein AoAbp1 (A. oryzae homolog of Saccharomyces cerevisiae Abp1p) as bait, a putative AAA (ATPases associated with diverse cellular activities) ATPase encoded by a gene termed aipA (AoAbp1 interacting protein) was identified. Further YTH analyses showed that the 346-370 amino-acid region of AipA interacts with the SH3 (Src homology 3) domains of AoAbp1. Moreover, AipA colocalized with AoAbp1 at the tip region, suggesting that AipA functions in endocytosis. Although aipA disruptants did not display defective growth, an aipA-overexpressing strain displayed impaired growth and wider hyphal morphology. In addition, we generated strains that overexpressed either aipA(K542A) or aipA(E596Q) , whose mutations were introduced into the AAA ATPase domain of AipA and would cause the defect of ATPase activity. In contrast to the aipA-overexpressing strain, neither aipA(K542A) - nor aipA(E596Q) -overexpressing strains showed defective growth. Moreover, only the aipA-overexpressing strain displayed a defect of FM4-64 transport to the Spitzenkörper, suggesting that AipA negatively regulates apical endocytic recycling and that the AAA ATPase domain of AipA is crucial for its function.