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So, Yee-Seul,Lee, Dong-Gi,Idnurm, Alexander,Ianiri, Giuseppe,Bahn Yong-Sun Genetics Society of America [etc.] 2019 Genetics Vol.212 No.4
<P> The target of rapamycin (TOR) pathway is an evolutionarily conserved signal transduction system that governs a plethora of eukaryotic biological processes, but its role in <I>Cryptococcus neoformans</I> remains elusive. In this study, we investigated the TOR pathway by functionally characterizing two Tor-like kinases, Tor1 and Tlk1, in <I>C. neoformans.</I> We successfully deleted <I>TLK1</I>, but not <I>TOR1. TLK1</I> deletion did not result in any evident <I>in vitro</I> phenotypes, suggesting that Tlk1 is dispensable for the growth of <I>C. neoformans.</I> We demonstrated that Tor1, but not Tlk1, is essential and the target of rapamycin by constructing and analyzing conditionally regulated strains and sporulation analysis of heterozygous mutants in the diploid strain background. To further analyze the Tor1 function, we constructed constitutive <I>TOR1</I> overexpression strains. Tor1 negatively regulated thermotolerance and the DNA damage response, which are two important virulence factors of <I>C. neoformans. TOR1</I> overexpression reduced Mpk1 phosphorylation, which is required for cell wall integrity and thermoresistance, and Rad53 phosphorylation, which governs the DNA damage response pathway. Tor1 is localized to the cytoplasm, but enriched in the vacuole membrane. Phosphoproteomics and transcriptomics revealed that Tor1 regulates a variety of biological processes, including metabolic processes, cytoskeleton organization, ribosome biogenesis, and stress response. TOR inhibition by rapamycin caused actin depolarization in a Tor1-dependent manner. Finally, screening rapamycin-sensitive and -resistant kinase and transcription factor mutants revealed that the TOR pathway may crosstalk with a number of stress signaling pathways. In conclusion, our study demonstrates that a single Tor1 kinase plays pleiotropic roles in <I>C. neoformans.</I> </P>
( Yee-seul So ),( Dong-hoon Yang ),( Kwang-woo Jung ),( Won-ki Huh ),( Yong-sun Bahn ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.2
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 aca1Δ::ACA1-GFP and aca1Δ::ACA1- GFP cac1Δ 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.