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Applied and Environmental Microbiology, October 2008, p. 6385-6396, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.01185-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Expression and Subcellular Localization of the Aflatoxin Pathway Enzyme Ver-1 Fused to Enhanced Green Fluorescent Protein {triangledown} ,{dagger}

Sung-Yong Hong1 and John E. Linz1,2,3,4*

Department of Food Science and Human Nutrition,1 National Food Safety and Toxicology Center,2 Department of Microbiology and Molecular Genetics,3 Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 488244

Received 28 May 2008/ Accepted 14 August 2008

Aflatoxin, a mycotoxin synthesized by Aspergillus spp., is among the most potent naturally occurring carcinogens known. Little is known about the subcellular organization of aflatoxin synthesis. Previously, we used transmission electron microscopy and immunogold labeling to demonstrate that the late aflatoxin enzyme OmtA localizes primarily to vacuoles in fungal cells on the substrate surface of colonies. In the present work, we monitored subcellular localization of Ver-1 in real time in living cells. Aspergillus parasiticus strain CS10-N2 was transformed with plasmid constructs that express enhanced green fluorescent protein (EGFP) fused to Ver-1. Analysis of transformants demonstrated that EGFP fused to Ver-1 at either the N or C terminus functionally complemented nonfunctional Ver-1 in recipient cells. Western blot analysis detected predominantly full-length Ver-1 fusion proteins in transformants. Confocal laser scanning microscopy demonstrated that Ver-1 fusion proteins localized in the cytoplasm and in the lumen of up to 80% of the vacuoles in hyphae grown for 48 h on solid media. Control EGFP (no Ver-1) expressed in transformants was observed in only 13% of the vacuoles at this time. These data support a model in which middle and late aflatoxin enzymes are synthesized in the cytoplasm and transported to vacuoles, where they participate in aflatoxin synthesis.

* Corresponding author. Mailing address: Department of Food Science and Human Nutrition, 234B GM Trout Building, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-8474. Fax: (517) 353-8963. E-mail: jlinz{at}msu.edu

{triangledown} Published ahead of print on 29 August 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, October 2008, p. 6385-6396, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.01185-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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