Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22897
標題: 以酵母菌雙雜交法分析冰花鹽誘導蛋白mcSKD1與mcSNF1和mcCPN1之間的交互作用
Yeast two-hybrid analysis of salt-induced mcSKD1 interacting with mcSNF1 and mcCPN1 in ice plant
作者: 林亞君
Lin, Ya-Chung
關鍵字: yeast two-hybrid;酵母菌雙雜交法;ice plant;protein-protein interaction;冰花;蛋白交互作用
出版社: 生命科學系所
引用: Alepuz, P.M., Cunningham, K.W., and Estruch, F. (1997). Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene. Mol. Microbiol. 26: 91-98. Ananieva, E.A., Gillaspy, G.E., Ely, A., Burnette, R.N., and Erickson, F.L. (2008). Interaction of the WD40 domain of a myoinositol polyphosphate 5-phosphatase with SnRK1 links inositol, sugar, and stress signaling. Plant Physiol. 148: 1868-1882. Andersson, U., Filipsson, K., Abbott, C.R., Woods, A., Smith, K., Bloom, S.R., Carling, D., and Small, C.J. (2004). AMP-activated protein kinase plays a role in the control of food intake. J. Biol. Chem. 279: 12005-12008. Babst, M., Wendland, B., Estepa, E.J., and Emr, S.D. (1998). The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17: 2982-2993. Bhalerao, R.P., Salchert, K., Bakó, L.,Ökrész, L., Szabados, L., Muranaka, T., Machida, Y., Schell, J., and Koncz, C. (1999). Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases. Proc. Natl. Acad. Sci. USA 96: 5322-5327. Bhatnagar, R.S., Ashrafi, K., Futterer, K., Waksman, G., and Gordon, J.I. (2001). Biology and enzymology of protein N-myristoylation. In The Enzymes, F. Tamanoi and D.S. Sigman, eds (San Diego, CA: Academic Press), pp. 241-286. Bohnert, H.J., and Cushman, J.C. (2000). The ice plant cometh: lessons in abiotic stress tolerance. J. Plant Growth Regul. 19: 334-346. Bohnert, H.J., Nelson, D.E., and Jensen, R.G. (1995). Adaptations to environmental stresses. Plant Cell 7: 1099-1111. Borden, K.L.B. (2000). RING domains: master builders of molecular scaffolds? J. Mol. Biol. 295: 1103-1112. Celenza, J.L., and Carlson, M. (1986). A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233: 1175-1180. Chen, Y.C. (2006). Identification of full-length sequence and analysis of protein domains of mcCPN1 gene in halophyte Mesembryanthemum crystallinum. Bachelor thesis, Department of Life Sciences, National Chung-Hsing University. Chen, Y.C. (2008). Analysis of gene expression and protein accumulation of an E3 ligase mcCPN1 in ice plant. Master thesis, Department of Life Sciences, National Chung-Hsing University. Dale, S., Arró, M., Becerra, B., Morrice, N.G., Boronat, A., Hardie, D.G., and Ferrer, A. (1995). Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (isoform HMGR1) from Arabidopsis thaliana, and its inactivation by phosphorylation at serine-577 by Brassica oleracea 3-hydroxy-3-methylglutaryl CoA reductase kinase. Eur. J. Biochem. 233: 506-513. Dougan, D.A., Mogk, A., Zeth, K., Turgay, K., and Bukau, B. (2002). AAA+ proteins and substrate recognition, it all depends on their partner in crime. FEBS Lett. 529: 6-10. Douglas, P., Pigaglio, E., Ferrer, A., Halford, N.G., and MacKintosh, C. (1997). Three spinach leaf nitrate reductase/3-hydroxy-3-methylglutaryl-CoA reductase kinases that are regulated by reversible phosphorylation and/or Ca2+ ions. Biochem. J. 325: 101-109. Farrás, R., Ferrando, A., Jásik, J., Kleinow, T., Ökrész, L., Tiburcio, A., Salchert, K., del Pozo, C., Schell, J., and Koncz, C. (2001). SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase. EMBO J. 20: 2742-2756. Frickey, T., and Lupas, A.N. (2004). Phylogenetic analysis of AAA proteins. J. Struct. Biol. 146: 2-10. Haas, T.J., Sliwinski, M.K., Martínez, D.E., Preuss, M., Ebine, K. Ueda, T. Nielsen, E., Odorizzi, G., and Otegui, M.S. (2007). The arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19: 1295-1312. Halford, N.G.., and Hardie, D.G. (1998). SNF1-related protein kinases: global regulators of carbon metabolism in plants? Plant Mol. Biol. 37: 735-748. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580. Hanson, P.I., and Whiteheart, S.W. (2005). AAA+ proteins: have engine, will work. Nature Rev. Mol. Cell Biol. 6: 519-529. Haro, R., Garciabeblas, B., and Rodriguez-Navarro, A. (1991). A novel P-type ATPase from yeast involved in sodium transport. FEBS Lett. 291: 189-191. Hislop, J.N., Marley, A., and von Zastrow, M. (2004). Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes. J. Biol. Chem. 279: 22522-22531. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G., and Jentsch, S. (2002). RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135-141. Hofmann, K., and Bucher, P. (1996). The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. Trends Biochem. Sci. 21:172-3. Hrabak, E.M,. Chan, C.W.M., Gribskov, M., Harper, J.F., Choi, J.H., Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R., Thomas, M., Walker-Simmons, K., Zhu, J.K., and Harmon, A.C. (2003). The arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132: 666-680. Huh, G.H., Damsz, B., Matsumoto, T.K., Reddy, M.P., Rus, A.M., Ibeas, J.I., Narasimhan, M.L., Bressan, R.A., Hasegawa, P.M. (2002). Salt causes ion disequilibrium-induced programmed cell death in yeast and plants. Plant J. 29: 649-659. Iyer, L.M., Leipe, D.D., Koonin, E.V., and Aravind, L. (2004). Evolutionary history and higher order classification of AAA+ ATPases. J. Struct. Biol. 146: 11-31. James, P., Halladay, J., and Craig, E.A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144: 1425-1436. Jefferies, R.L. (1981). Osmotic adjustment and the response of halophytic plants to salinity. Bio. Sci. 31: 42-46. Jiang, R., and Carlson, M. (1996). Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes Dev. 10: 3105-3115. Jou, Y.T. (2007). Characterization of bladder cell-specific mcSKD1 and its interacting protein in halophyte Mesembryanthemum crystallinum L. Ph.D. thesis, Department of Life Sciences, National Chung-Hsing University Jou,Y.T., Chiang, C.P., Jauh, G.Y. ,and Yen, H.E. (2006). Functional characterization of ice plant SKD1, an AAA-type ATPase associated with the endoplasmic reticulum-golgi network, and its role in adaptation to salt stress. Plant Physiol. 141: 135-146. Jou, Y.T., Chou, P.H., He, M.C., Hung, Y.H., and Yen, H.E. (2004). Tissue-specific expression and functional complementation of a yeast potassium-uptake mutant by a salt-induced ice plant gene mcSKD1. Plant Mol. Biol. 54: 881-893. Lee, J.O., Rieu, P., Arnaout, M.A., and Liddington, R. (1995). Crystal structure of the A domain from the α subunit of integrin CR3 (CD11b/CD18). Cell 80: 631-638. Mazzucotelli, E., Belloni, S., Marone, D., De Leonardis, A.M., Guerra, D., Di Fonzo, N., Cattivelli, L., and Mastrangelo, A.M. (2006). The E3 ubiquitin ligase gene family in plants: regulation by degradation. Curr. Genomics 7: 509-522. Périer, F., Coulter, K.L., Liang, H., Radeke, C.M., Gaber, R.F., and Vandenberg, C.A. (1994). Identification of a novel mammalian member of the NSF/CDC48p/Paslp/TBP-1 family through heterologous expression in yeast. FEBS Lett. 351: 286-290. Ponting, C.P., Aravind, L., Schultz, J., Bork, P., and Koonin, E.V. (1999). Eukaryotic signaling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J. Mol. Biol. 289: 729-745. Rankin, C.A., Joazeiro, C.A., Floor, E. and Hunter, T. (2001). E3 ubiquitinprotein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements. J. Biomed. Sci. 8: 421-429. Scott, A., Gaspar, J., Stuchell-Brereton, M.D., Alam, S.L., Skalicky, J.J., and Sundquist, W.I. (2005). Structure and ESCRT-III protein interactions of the MIT domain of human VPS4A. Proc. Natl. Acad. Sci. USA 102: 13813-13818. Shieh, S.S. (2005). Functional analysis of atSKD1 gene in arabidopsis under high salinity by gene silencing. Master thesis, Department of Life Sciences, National Chung-Hsing University. Spitzer, C., Reyes, F.C., Buono, R., Sliwinski, M.K., Haas, T.J., and Otegui, M.S. (2009). The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in arabidopsis and are required for plant development. Plant Cell 21: 749-766. Stone, S.L., Hauksdóttir, H., Troy, A., Herschleb, J., Kraft, E., and Callis, J. (2005). Functional analysis of the RING-type ubiquitin ligase family of arabidopsis. Plant Physiol. 137: 13-30. Tassan, J.P., and Le Goff, X. (2004). An overview of the KIN1/PAR-1/MARK kinase family. Biol. Cell 96:193-199. Tuckwell, D. (1999). Evolution of von Willebrand factor A (VWA) domains. Biochem. Soc. Trans. 27: 835-840. Vernon, D.M., and Bohnert, H.J. (1992). A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum. EMBO J. 11: 2077-2085. Wang, Y.C. (2006). Isolation and characterization of SNF-1 gene in halophyte Mesembryanthemum crystallinum L. Bachelor thesis, Department of Life Sciences, National Chung-Hsing University. Ward, J.M., Hirschi, K.D., and Sze, H. (2003). Plants pass the salt. Trends Plant Sci. 8: 200-201. Whittaker, C.A., and Hynes, R.O. (2002). Distribution and evolution of von Willebrand/Integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol. Biol. Cell 13: 3369-3387. Yang, F.Y. (2007). Analyses of gene expression and protein accumulation of a signal transduction-related mcSNF1 in halophyte Mesembryanthemum crystallinum under salt stress. Master thesis, Department of Life Sciences, National Chung-Hsing University. Yen, H.E., Wu, S.M., Hung, Y.H., and Yen, S.K. (2000). Isolation of 3 salt-induced low-abundance cDNAs from light-grown callus of Mesembryanthemum crystallinum by suppression subtractive hybridization. Physiol. Plant. 110: 402-409. Yeo, S.C., Xu, L., Ren, J., Boulton, V.J., Wagle, M.D., Liu, C., Ren, G., Wong, P., Zahn, R., and Sasajala, P. (2003). Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae. J. Cell Sci. 116: 3957-3970. Yin, X.J., Volk, S., Ljung, K., Mehlmer, N., Dolezal, K., Ditengou, F., Hanano, S., Davis, S.J., Schmelzer, E., Sandberg, G., Teige, M., Palme, K., Pickart, C., and Bachmaira, A. (2007). Ubiquitin lysine 63 chain-forming ligases regulate apical dominance in arabidopsis. Plant Cell 19: 1898-1911. Zhang, J., Kong, C., Xie, H., McPherson, P.S., Grinstein, S. and Trimble, W.S. (1999). Phosphatidylinositol polyphosphate binding to the mammalian septin H5 is modulated by GTP. Curr. Biol. 9: 1458-1467.
摘要: 
冰花(Mesembryanthemum crystallinum L.)為耐鹽機制的模式植物,常用於探討鹽分逆境下植物所誘發的生理反應,mcSKD1 (suppressor of K+ transport growth defect 1)為冰花一鹽誘導蛋白,具有三個保留區(conserved domain),microtubule interacting and trafficking molecule domain (MIT)、ATPase associated with a variety of cellular activities (AAA)和Vps4 C terminal oligomerisation domain (Vps4_C),與酵母菌中內膜系統的蛋白運送相關的Vps4 (vacuolar protein sorting 4)同源,而mcSKD1分佈於冰花細胞的內質網(endoplasmic reticulum)和高基氏體(Golgi),推測mcSKD1可能參與冰花的內膜系統蛋白運送機制。前人研究已利用酵母菌雙雜交系統篩選出和mcSKD1具有交互作用之蛋白mcSNF1 (sucrose non-fermenting 1)與mcCPN1 (copine 1)。mcSNF1與酵母菌中調控碳代謝相關酵素的SNF1同源,具有三個conserved domains,由N端至C端依序為serine/threonine protein kinases domain (S/T)、ubiquitin-associated domain (UBA)和kinase-associated domain 1 (KA1)。mcCPN1的N端為具有myristoylation修飾位置的Front domain、von Willebrand factor type A domain (vWA)和C端的really interesting new gene domain (RING),之前研究中確定mcCPN1具有E3 ligase的活性,能夠將mcSKD1接上ubiquitin。然而上述三蛋白之間產生交互作用的位置及作用強度仍是未知,故本論文中即利用酵母菌雙雜交法分析三者之間的交互作用關係。將各個全長和domain的序列分別構築於表現載體後轉入酵母菌中,使產生48種的待測組合,利用營養篩選培養基以連續稀釋法進行生長分析,並配合β-galactosidase酵素活性分析,區別三者之間的交互作用的高低。結果顯示,mcSKD1以MIT和Vps4_C domain與mcSNF1的KA1 domain產生極高度的交互作用;mcSKD1同時以MIT和AAA domain與mcCPN1的vWA domain產生高度的交互作用;mcCPN1以Front和RING domain與mcSNF1的KA1 domain產生極高度的交互作用。推測mcSNF1為mcSKD1和mcCPN1之間的媒介蛋白,以KA1 domain將mcSKD1和mcCPN1兩者拉近,使三者交互作用關係更為緊密,而藉由調控內膜系統的蛋白運送以及ubiquitination相關途徑參與植物在鹽逆境下的生理反應。

Ice plant (Mesembryanthemum crystallinum L.) is a model plant for study salt-induced physiological responses. In ice plant, mcSKD1 (suppressor of K+ transport growth defect 1) is a salt-induced protein, it contains three conserved domains, microtubule interacting and trafficking molecule domain (MIT), ATPase associated with a variety of cellular activities (AAA), and Vps4 C terminal oligomerisation domain (Vps4_C). McSKD1 share homology to yeast Vps4 (vacuolar protein sorting 4) which involves in vesicle trafficking mechanism, and mcSKD1 also locates in endoplasmic reticulum to Golgi network. Thus, mcSKD1 may participate in protein sorting mechanism. McSNF1 and mcCPN1 were indentified interacting with mcSKD1 by yeast two-hybrid system in previous study. McSNF1 share homology to yeast SNF1 which involves in carbon metabolism pathway, and it contains three conserved domains, serine/threonine protein kinases domain (S/T), ubiquitin-associated domain (UBA), and kinase-associated domain 1 (KA1). McCPN1 contains Front domain, von Willebrand factor type A domain (vWA), and really interesting new gene domain (RING). In previous study, it has been shown that mcCPN1 has E3 ligase activity and conjugates ubiquitin chain to mcSKD1. However, the interaction regions and degree of interaction between mcSKD1, mcSNF1, and mcCPN1 are still unknown. In this study, I use yeast two-hybrid to analyze the interaction network among these three proteins. The full-length genes and domain fragments were constructed into expressing vectors, and transformed into yeast cells, respectively. I obtained 48 different combinations, and analyzed them with serial dilution assay and β-galactosidase assay to distinguish the interaction levels between mcSKD1, mcSNF1, and mcCPN1. The results showed that MIT, Vps4_C, and KA1 domains are the regions of very high degree of interaction between mcSKD1 and mcSNF1; MIT, AAA, and vWA domains are the regions of high degree of interaction between mcSKD1 and mcCPN1; Front, RING, and KA1 domains are the regions of very high degree of interaction between mcCPN1 and mcSNF1. Those results suggested that the KA1 domain of mcSNF1 mediates the interaction between mcSKD1 and mcCPN1, and brought the protein complex together to function in protein sorting and ubiquitination pathway under salt stress.
URI: http://hdl.handle.net/11455/22897
其他識別: U0005-1607200917182600
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