Molecular characterization of adenylyl cyclase complex proteins using versatile protein-tagging plasmid systems in Cryptococcus neoformans

Yee Seul So, Dong Hoon Yang, Kwang Woo Jung, Won Ki Huh, Yong-Sun Bahn

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 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 non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, 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∆::ACA1-GFP and aca1∆::ACA1-GFP cac1∆ 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.

Original languageEnglish
Pages (from-to)357-364
Number of pages8
JournalJournal of microbiology and biotechnology
Volume27
Issue number2
DOIs
Publication statusPublished - 2017 Feb 1

Fingerprint

Cryptococcus neoformans
Adenylyl Cyclases
Plasmids
Streptothricins
Proteins
Neomycin
Immunoprecipitation
Organelles
Virulence
Mass Spectrometry
Fluorescence
Cell Membrane

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

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title = "Molecular characterization of adenylyl cyclase complex proteins using versatile protein-tagging plasmid systems in Cryptococcus 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 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 non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, 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∆::ACA1-GFP and aca1∆::ACA1-GFP cac1∆ 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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Molecular characterization of adenylyl cyclase complex proteins using versatile protein-tagging plasmid systems in Cryptococcus neoformans. / So, Yee Seul; Yang, Dong Hoon; Jung, Kwang Woo; Huh, Won Ki; Bahn, Yong-Sun.

In: Journal of microbiology and biotechnology, Vol. 27, No. 2, 01.02.2017, p. 357-364.

Research output: Contribution to journalArticle

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AU - So, Yee Seul

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AU - Jung, Kwang Woo

AU - Huh, Won Ki

AU - Bahn, Yong-Sun

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