KMID : 0545120170270020357
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Journal of Microbiology and Biotechnology 2017 Volume.27 No. 2 p.357 ~ p.364
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Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus neoformans
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So Yee-Seul
Yang Dong-Hoon Jung Kwang-Woo Huh Won-Ki Bahn Yong-Sun
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Abstract
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In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, 4¡¿FLAG, or 6¡¿HA, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and nonnuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by coimmunoprecipitation, using Cac1-6¡¿HA and Aca1-4¡¿FLAG tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using aca1¥Ä::ACA1-GFP and aca1¥Ä::ACA1- GFP cac1¥Ä strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.
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KEYWORD
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Cyclic AMP, adenylyl cyclase-associated protein, Cac1, Aca1
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