We explored novel components involved in endocytosis by the yeast two-hybrid (YTH) screening using AoAbp1 (Aspergillus oryzae actin binding protein) as bait. A gene named aipA (AoAbp1 interacting protein) which encodes a putative AAA (ATPases associated with diverse cellular activities) ATPase was obtained. Further YTH analyses showed that 346-370 aa region of AipA interacted with the two SH3 domains of AoAbp1. AipA interacted with AoAbp1 in vitro, and in A. oryzae EGFP-AipA co-localized with AoAbp1-mDsRed at the tip region, suggesting that AipA functions in endocytosis. Although aipA disruptants did not display any phenotypic alteration in several culture conditions, aipA-overexpressing strains showed defective growth and the aberrant hyphal morphology. Moreover, we generated strains which have mutations of either aipAK542A or aipAE596Q. These mutations were introduced in the ATPase domain of AipA and would cause defect in the ATPase activity. In contrast to the strain overexpressing WT aipA, the growth of mutated strains was normal, suggesting that ATPase activity is important for the function of AipA. Furthermore, the aipA-overexpressing strain displayed a delay in FM4- 64 transport to Spitzenkorper, whereas the mutated aipA-overexpressing strains did not, suggesting that AipA negatively regulates apical endocytic recycling.
Full conference title:
26th Fungal Genetics Conference
- Fungal Genetics Conference 26th (2005)