| Research Abstract |
In yeast S. cerevisiae, vacuoles play very important roles in pH and osmotic regulation, protein degradation and storage of amino acids, small ions as well as polyphosphates. In filamentous fungi, however, little is known about vacuolar functions at a molecular level. Several genes homologous to yeast VPS (VPS1, VPS45, and VPS21) and VAM (VAM4, and VAM6) genes were isolated from Aspergillus nidulans and designated the genes as vpsA, vpsB, vpsC, avaA, and avaB, respectively. These gene disruption experiments of A. nidulans showed poor growth and contained fragmented vacuoles. These results suggested that the genes isolated are involved in vacuolar biogenesis and functions. In addition, cpyA (carboxy peptidase Y homologue) gene was also isolated as a marker enzyme of vacuoles from A. nidulans. The cpyA gene was fused with EGFP (Enhanced Green Fluorescent Protein) gene and the resulting fusion gene was introduced into A. nidulans and A. oryzae, respectively. Vacuoles are clearly visualized by a fluorescent microscope in the living fungi by expressing of CPY-EGFP. Eighteen hfc (hyper EGFP fluoresce in conidia) mutants, 20 dfc (darker fluorescence in conidia) mutants, and 22 hfm (hyper EGFP fluorescence in the medium) mutants were isolated from A. oryzae expressing CPY-EGFP as a parent strain using a FACS ( fluorescence activated cell sorter) or a fluorescent micro plate reader. hfc-1 mutant showed very poor growth in alkaline medium. Fragmented vacuoles, which are similar to class B vps mutants in yeast, were observed in hfc-3, hfc-4, dfc-14, and hfm-4 mutants. Accumulation of small spherical structure near vacuolar membrane was observed in dfc-14, and hfm-4 mutants in alkaline medium, which are similar to class E vps mutants.
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