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http://dx.doi.org/10.4014/jmb.1609.09036

Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus neoformans  

So, Yee-Seul (Department of Biotechnology, Yonsei University)
Yang, Dong-Hoon (Department of Biotechnology, Yonsei University)
Jung, Kwang-Woo (Department of Biotechnology, Yonsei University)
Huh, Won-Ki (Department of Biological Sciences, Seoul National University)
Bahn, Yong-Sun (Department of Biotechnology, Yonsei University)
Publication Information
Journal of Microbiology and Biotechnology / v.27, no.2, 2017 , pp. 357-364 More about this Journal
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, $4{\times}FLAG$, or $6{\times}HA$, 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 $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ 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.
Keywords
Cyclic AMP; adenylyl cyclase-associated protein; Cac1; Aca1;
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1 Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM. 2009. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23: 525-530.   DOI
2 Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, et al. 2010. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin. Infect. Dis. 50: 291-322.   DOI
3 Davidson RC, Blankenship JR, Kraus PR, de Jesus Berrios M, Hull CM, D'Souza C, et al. 2002. A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148: 2607-2615.   DOI
4 Kim MS, Kim SY, Yoon JK, Lee YW, Bahn YS. 2009. An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with nat-split markers. Biochem. Biophys. Res. Commun. 390: 983-988.   DOI
5 Davidson RC, Cruz MC, Sia RA, Allen B, Alspaugh JA, Heitman J. 2000. Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genet. Biol. 29: 38-48.   DOI
6 Ory JJ, Griffith CL, Doering TL. 2004. An efficiently regulated promoter system for Cryptococcus neoformans utilizing the ctr4 promoter. Yeast 21: 919-926.   DOI
7 Idnurm A, Reedy JL, Nussbaum JC, Heitman J. 2004. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot. Cell 3: 420-429.   DOI
8 Liu OW, Chun CD, Chow ED, Chen C, Madhani HD, Noble SM. 2008. Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135: 174-188.   DOI
9 Baker LG, Lodge JK. 2012. Multiple gene deletion in Cryptococcus neoformans using the cre-lox system. Methods Mol. Biol. 845: 85-98.
10 Bahn YS, Jung KW. 2013. Stress signaling pathways for the pathogenicity of Cryptococcus. Eukaryot. Cell 12: 1564-1577.   DOI
11 Habeler G, Natter K, Thallinger GG, Crawford ME, Kohlwein SD, Trajanoski Z. 2002. Ypl.Db: the yeast protein localization database. Nucleic Acids Res. 30: 80-83.   DOI
12 Alspaugh JA, Pukkila-Worley R, Harashima T, Cavallo LM, Funnell D, Cox GM, et al. 2002. Adenylyl cyclase functions downstream of the $G{\alpha}$ protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans. Eukaryot. Cell 1: 75-84.   DOI
13 Bahn YS, Hicks JK, Giles SS, Cox GM, Heitman J. 2004. Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade. Eukaryot. Cell 3: 1476-1491.   DOI
14 Maeng S, Ko YJ, Kim GB, Jung KW, Floyd A, Heitman J, et al. 2010. Comparative transcriptome analysis reveals novel roles of the Ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans. Eukaryot. Cell 9: 360-378.   DOI
15 Jung KW, Strain AK, Nielsen K, Jung KH, Bahn YS. 2012. Two cation transporters Ena1 and Nha1 cooperatively modulate ion homeostasis, antifungal drug resistance, and virulence of Cryptococcus neoformans via the HOG pathway. Fungal. Genet. Biol. 49: 332-345.   DOI
16 Lee KT, So YS, Yang DH, Jung KW, Choi J, Lee DG, et al. 2016. Systematic fungal analysis of kinases in the fungal pathogen Cryptococcus neoformans. Nat. Commun. 7: 12766.   DOI
17 Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20: 87-90.   DOI
18 Hubberstey AV, Mottillo EP. 2002. Cyclase-associated proteins: capacity for linking signal transduction and actin polymerization. FASEB J. 16: 487-499.   DOI
19 Rodaway AR, Sternberg MJ, Bentley DL. 1989. Similarity in membrane proteins. Nature 342: 624.
20 Willmund F, del Alamo M, Pechmann S, Chen T, Albanese V, Dammer EB, et al. 2013. The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis. Cell 152: 196-209.   DOI
21 Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, et al. 2002. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415: 141-147.   DOI
22 Chang YC, Lamichhane AK, Kwon-Chung KJ. 2012. Role of actin-bundling protein Sac6 in growth of Cryptococcus neoformans at low oxygen concentration. Eukaryot. Cell 11: 943-951.   DOI
23 Tarassov K, Messier V, Landry CR, Radinovic S, Serna Molina MM, Shames I, et al. 2008. An in vivo map of the yeast protein interactome. Science 320: 1465-1470.   DOI
24 Landgraf C, Panni S, Montecchi-Palazzi L, Castagnoli L, Schneider-Mergener J, Volkmer-Engert R, et al. 2004. Protein interaction networks by proteome peptide scanning. PLoS Biol. 2: E14.   DOI
25 Mitchell SF, Jain S, She M, Parker R. 2013. Global analysis of yeast mRNPs. Nat. Struct. Mol. Biol. 20: 127-133.   DOI
26 Panepinto J , Liu L, Ramos J , Zhu X, Valyi-Nagy T, Eksi S, et al. 2005. The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans. J. Clin. Invest. 115: 632-641.   DOI
27 Quintero-Monzon O, Jonasson EM, Bertling E, Talarico L, Chaudhry F, Sihvo M, et al. 2009. Reconstitution and dissection of the 600-kDa Srv2/CAP complex: roles for oligomerization and cofilin-actin binding in driving actin turnover. J. Biol. Chem. 284: 10923-10934.   DOI
28 Gilmore JM, Sardiu ME, Venkatesh S, Stutzman B, Peak A, Seidel CW, et al. 2012. Characterization of a highly conserved histone related protein, Ydl156w, and its functional associations using quantitative proteomic analyses. Mol. Cell Proteomics 11: M111.011544.   DOI
29 Wang Y, Shen G, Gong J, Shen D, Whittington A, Qing J, et al. 2014. Noncanonical $G{\beta}$ Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans. J. Biol. Chem. 289: 12202-12216.   DOI
30 Shima F, Okada T, Kido M, Sen H, Tanaka Y, Tamada M, et al. 2000. Association of yeast adenylyl cyclase with cyclaseassociated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation. Mol. Cell. Biol. 20: 26-33.   DOI
31 Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, et al. 2002. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415: 180-183.   DOI