title: Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems inCryptococcus neoformans.
abstract: 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 pathogenCryptococcus 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 ofC. 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 usingaca1Δ::ACA1-GFPandaca1Δ::ACA1- GFP cac1Δstrains 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 inC. neoformansand further our understanding of its biology.
authors: Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.
authors: Yee-Seul
authors: YS
authors: So
authors: Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.
authors: Dong-Hoon
authors: DH
authors: Yang
authors: Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.
authors: Kwang-Woo
authors: KW
authors: Jung
authors: Department of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea.
authors: Won-Ki
authors: WK
authors: Huh
authors: Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.
authors: Yong-Sun
authors: YS
authors: Bahn
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002934:Cinnamates / Q000494:pharmacology; D003455:Cryptococcus neoformans / Q000235:genetics* / Q000502:physiology; D000242:Cyclic AMP / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D005800:Genes, Fungal; D005818:Genetic Engineering / Q000379:methods; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D013058:Mass Spectrometry; D010957:Plasmids; D015398:Signal Transduction
chemical_list: D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A; D000242:Cyclic AMP; D000262:Adenylyl Cyclases
keywords: Aca1; Cac1; Cyclic AMP; adenylyl cyclase-associated protein