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Applied and Environmental Microbiology, August 2005, p. 4856-4861, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4856-4861.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

Mamoru Ohneda, Manabu Arioka, and Katsuhiko Kitamoto*

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 27 August 2004/ Accepted 11 March 2005

The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22 hfm (hyper-EGFP fluorescence released into the medium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The dfc and hfm mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in Saccharomyces cerevisiae vps mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.

* Corresponding author. Mailing address: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81-3-5841-5161. Fax: 81-3-5841-8033. E-mail: akitamo{at}mail.ecc.u-tokyo.ac.jp.

Applied and Environmental Microbiology, August 2005, p. 4856-4861, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4856-4861.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Shoji, J.-y., Arioka, M., Kitamoto, K. (2006). Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae. Eukaryot Cell 5: 411-421 [Abstract] [Full Text]  


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