Abstract
Similar to the activities of transcription factors (TFs) in other eukaryotes, activities of many plant TFs are determined via regulated proteolysis by the ubiquitin/26S proteasome system. Thus, to fully understand the function of a TF, it is important to determine the fate of the active TF protein and unravel the environmental and intrinsic signals that control its total cellular level. Here we describe how to determine whether a TF of interest is targeted to the 26S proteasome for degradation. The given method combines analyses of the effects of translational inhibition and the inhibition of proteasome activity. An important requirement for these experiments is to monitor in parallel the effects of translational and proteasomal inhibition on the abundance of the TF and (1) on ubiquitin, which becomes rapidly depleted upon translational inhibition (2), on polyubiquitinated proteins, which accumulate upon successful inhibition of the 26S proteasome, and (3) on glutamine synthase, a very stable protein that is used as a general metabolic control. The method described here can be used to test TF stability under a variety of conditions and in different genetic backgrounds.
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References
Greenbaum, D., Colangelo, C., Williams, K., and Gerstein, M. (2003) Comparing protein abundance and mRNA expression levels on a genomic scale. Genome Biol. 4, 117.
Kislinger, T., Cox, B., Kannan, A., Chung, C., Hu, P., Ignatchenko, A., Scott, M. S., Gramolini, A. O., Morris, Q., Hallett, M. T., Rossant, J., Hughes, T. R., Frey, B., and Emili, A. (2006) Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling. Cell 125, 173–186.
Fu, N., Drinnenberg, I., Kelso, J., Wu, J.-R., Pääbo, S., Zeng, R., and Khaitovich, P. (2007) Comparison of protein and mRNA expression evolution in humans and chimpanzees. PLoS One 2, e216.
Nie, L., Wu, G., Culley, D. E., Scholten, J. C., and Zhang, W. (2007) Integrative analysis of transcriptomic and proteomic data: challenges, solutions and applications. Crit. Rev. Biotechnol. 27, 63–75.
Gagne, J. M., Smalle, J., Gingerich, D. J., Walker, J. M., Yoo, S. D., Yanagisawa, S., and Vierstra, R. D. (2004) Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation. Proc. Natl. Acad. Sci. USA 101, 6803–6808.
Guo, H., and Ecker, J. R. (2003) Plant responses to ethylene gas are mediated by SCFEBF1/EBF2-dependent proteolysis of EIN3 transcription factor. Cell 115, 667–677.
Potuschak, T., Lechner, E., Parmentier, Y., Yanagisawa, S., Grava, S., Koncz, C., and Genschik, P. (2003) EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins: EBF1 and EBF2. Cell 115, 679–689.
Schwager, K. M., Calderon-Villalobos, L. I., Dohmann, E. M., Willige, B. C., Knierer, S., Nill, C., and Schwechheimer, C. (2007) Characterization of the VIER F-BOX PROTEINE genes from Arabidopsis reveals their importance for plant growth and development. Plant Cell 19, 1163–1178.
Osterlund, M. T., Hardtke, C. S., Wei, N., and Deng, X. W. (2000) Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature 405, 462–466.
Gray, W. M., Kepinski, S., Rouse, D., Leyser, O., and Estelle, M. (2001) Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature 414, 271–276.
Kepinski, S., and Leyser, O. (2004) Auxin-induced SCFTIR1-Aux/IAA interaction involves stable modification of the SCFTIR1 complex. Proc. Natl. Acad. Sci. USA 101, 12381–12386.
Yang, X., Lee, S., So, J. H., Dharmasiri, S., Dharmasiri, N., Ge, L., Jensen, C., Hangarter, R., Hobbie, L., and Estelle, M. (2004) The IAA1 protein is encoded by AXR5 and is a substrate of SCFTIR1. Plant J. 40, 772–782.
Zenser, N., Ellsmore, A., Leasure, C., and Callis, J. (2001) Auxin modulates the degradation rate of Aux/IAA proteins. Proc. Natl. Acad. Sci. USA 98, 11795–11800.
Chini, A., Fonseca, S., Fernández, G., Adie, B., Chico, J. M., Lorenzo, O., GarcÃa-Casado, G., López-Vidriero, I., Lozano, F. M., Ponce, M. R., Micol, J. L., and Solano, R. (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448, 666–671.
He, J. X., Gendron, J. M., Yang, Y., Li, J., and Wang, Z. Y. (2002) The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis. Proc. Natl. Acad. Sci. USA 99, 10185–10190.
Borissenko, L., and Groll, M. (2007) Diversity of proteasomal missions: fine tuning of the immune response. Biol. Chem. 388, 947–955.
DeMartino, G. N., and Gillette, T. G. (2007) Proteasomes: machines for all reasons. Cell 129, 659–662.
Kurepa, J., Toh-e, A., and Smalle, J. A. (2008) 26S proteasome regulatory particle mutants have increased oxidative stress tolerance. Plant J. 53, 102–114.
Hanna, J., and Finley, D. (2007) A proteasome for all occasions. FEBS Lett. 581, 2854–2861.
Smalle, J. A., and Vierstra, R. D. (2004) The ubiquitin 26S proteasome proteolytic pathway. Annu. Rev. Plant Biol. 55, 555–590.
Dreher, K., and Callis, J. (2007) Ubiquitin, hormones and biotic stress in plants. Ann. Bot. (Lond.) 99, 787–822.
Crews, C. M. (2003) Feeding the machine: mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins. Curr. Opin. Chem. Biol. 7, 534–539.
Pickart, C. M. (2000) Ubiquitin in chains. Trends Biochem. Sci. 25, 544–548.
Thrower, J. S., Hoffman, L., Rechsteiner, M., and Pickart, C. M. (2000) Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102.
Kurepa, J., Karangwa, C., Duke, L. S., and Smalle, J. A. (2010) Arabidopsis sensitivity to protein synthesis inhibitors depends on 26S proteasome activity. Plant Cell Rep. 29, 249–259.
Kurepa, J., and Smalle, J. A. (2008) Structure, function and regulation of plant proteasomes. Biochimie 90, 324–335.
Swerdlow, P. S., Finley, D., and Varshavsky, A. (1986) Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal. Biochem. 156, 147–153.
Acknowledgments
This work was supported by the KTRD Center in Lexington, KY, and by grants from NSF (# 0919991) and from the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service (#2005-35304-16043).
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Kurepa, J., Smalle, J.A. (2011). Assaying Transcription Factor Stability. In: Yuan, L., Perry, S. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 754. Humana Press. https://doi.org/10.1007/978-1-61779-154-3_12
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DOI: https://doi.org/10.1007/978-1-61779-154-3_12
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