The goal of this project is to describe the network of Arabidopsis proteins responsible for the covalent attachment of ubiquitin (Ub) and to develop research tools for their analysis. The post-translational conjugation of one or more Ubs to selected intracellular proteins plays an integral role in numerous growth, developmental, and metabolic processes in plants via its ability to modify the function and/or half-life of its targets. Whereas its main function is to commit proteins for degradation by the 26S proteasome, other functions have become apparent more recently, including roles in DNA repair, lysosomal catabolism, intracellular trafficking, and the regulation of transcription. Among the enzymes responsible for Ub conjugation, the Ub-protein ligases (or E3s) are the crucial elements that control both target selectivity and the nature of the Ub linkage. Arabidopsis appears to contain over 1,300 E3 components, making this collection one of the largest functional groups in this plant (~5% of the proteome). This project is directed toward defining the E3 families and their targets in Arabidopsis, using the large battery of methods, reagents and mutants assembled during a previous award. The Arabidopsis E3 protein families will be further annotated and their biochemical activities will be defined. Potential functions will be explored by expression studies with DNA microarrays and by localization using GFP-E3 fusions. Interaction strategies (yeast-two-hybrid and co-immunoprecipitation) using individual E3s as bait will be exploited to identify potential targets and various accessory factors. High-throughput protein microarrays will identify targets, using individual E3s or their target-recognition modules as probes. The processes controlled by ubiquitination will be explored by the phenotypic analysis in bulk of large collections of E2 and E3 mutants. Affinity approaches coupled with tagged Ubs will be employed to isolate ubiquitinated proteins from Arabidopsis. These conjugates will then be identified by various mass spectrometric techniques to provide an extensive library of Ub targets. And finally, the web-accessible PlantsUBQ database (http://plantsubq.genomics.purdue.edu) of genes, mutants, targets, and other information pertinent to ubiquitination will be expanded for broad scientific exposure.
Broader Impact: The results generated by this study will form an essential framework for understanding E3 diversity, help reveal specific functions for each E3 type, and likely assign function to a number of uncharacterized Arabidoposis genes, thus helping define the genome of this plant. Ultimately, the results will provide comprehensive information on the role of this post-translational modification in plants that ultimately can be used to devise new strategies to improve crop productivity. In addition to the training of graduate students and postdoctoral fellows, a main educational goal will be the inclusion of minority undergraduates in the discovery process as part of a summer research program.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH Next
(Showing: 1 - 20 of 26).
Binder, BM; Walker, JM; Gagne, JM; Emborg, TJ; Hemmann, G; Bleecker, AB; Vierstra, RD.
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PLANT CELL,
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Book, AJ, Yang, P, Scalf, M, Smith, LM, Vierstra, RD.
"Tripeptidyl peptidase II. An oligomeric protease complex from Arabidopsis.,"
Plant Physiol,
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2005,
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Chen, HD; Shen, YP; Tang, XB; Yu, L; Wang, J; Guo, L; Zhang, Y; Zhang, HY; Feng, SH; Strickland, E; Zheng, N; Deng, XW.
"Arabidopsis CULLIN4 forms an E3 ubiquitin ligase with RBX1 and the CDD complex in mediating light control of development,"
PLANT CELL,
v.18,
2006,
p. 1991
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Dharmasiri, N., Dharmasiri, S., and M. Estelle.
"The F-box protein TIR1 is an auxin receptor,"
Nature,
v.435,
2005,
p. 441.
Dharmasiri, N., Dharmasiri, S., Weijers, D., Lechner. E., Yamada. M., Hobbie. L., Ehrismann. J.S., Jürgens, G., and Estelle, M.
"Plant development is regulated by a family of auxin receptor F-box proteins.,"
Dev Cell,
v.9,
2005,
p. 109.
Dharmasiri, N; Dharmasiri, S; Weijers, D; Karunarathna, N; Jurgens, G; Estelle, M.
"AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis,"
PLANT JOURNAL,
v.52,
2007,
p. 114
- 123.
Downes, BP; Saracco, SA; Lee, SS; Crowell, DN; Vierstra, RD.
"MUBs, a family of ubiquitin-fold proteins that are plasma membrane-anchored by prenylation,"
JOURNAL OF BIOLOGICAL CHEMISTRY,
v.281,
2006,
p. 27145
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Figueroa, P., Gusmaroli, G., Serino, G., Habashi, J., Ma, L., Shen, Y., Feng, S., Bostick, M., Callis, J., Hellmann, H., and Deng, X.W.
"Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development
and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase
complexes in vivo,"
Plant Cell,
v.17,
2005,
p. 1180.
Gingerich DJ, Gagne JM, Salter DW, Hellmann H, Estelle M, Ma L, Vierstra RD.
"Cullins 3a and 3b Assemble with Members of the Broad Complex/Tramtrack/Bric-a-Brac
(BTB) Protein Family to Form Essential Ubiquitin-Protein Ligases (E3s) in Arabidopsis,"
J Biol. Chem,
v.280,
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Gingerich, DJ; Hanada, K; Shiu, SH; Vierstra, RD.
"Large-scale, lineage-specific expansion of a bric-a-brac/tramtrack/broad complex ubiquitin-ligase gene family in rice,"
PLANT CELL,
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Gong W, He K, Covington M, Dinesh-Kumar SP., Snyder M, Harmer SL, Zhu YX and X.W. Deng.
"The development of protein microarrays and their applications in DNA?protein and protein?protein interaction analyses of Arabidopsis transcription factors,"
Mol. Plant,
v.1,
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Gusmaroli, G; Figueroa, P; Serino, G; Deng, XW.
"Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis cullin3-based E3 ligases,"
PLANT CELL,
v.19,
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p. 564
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Johnson, LM; Bostick, M; Zhang, XY; Kraft, E; Henderson, I; Callis, J; Jacobsen, SE.
"The SRA methyl-cytosine-binding domain links DNA and histone methylation,"
CURRENT BIOLOGY,
v.17,
2007,
p. 379
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Kraft, E., Stone, S. L., Ma, L., Su, N., Gao, Y., Lau, O. S., Deng, X. W., and Callis, J..
"Genome analysis and functional characterization of the E2 and RING-type E3 ligase
ubiquitination enzymes of Arabidopsis,"
Plant Physiol,
v.139,
2005,
p. 1597.
Lee JH, Terzaghi W, Gusmaroli G, Charron JB, Yoon HJ, Chen H, He YJ, Xiong Y, and X.W. Deng.
"Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases,"
Plant Cell,
v.20,
2008,
p. 152.
Moon, J; Zhao, YD; Dai, XH; Zhang, WJ; Gray, WM; Huq, E; Estelle, M.
"A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis,"
PLANT PHYSIOLOGY,
v.143,
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Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O., Jones, J. D.G.
"An Inducible MiRNA Contributes to Arabidopsis Basal Resistance by Repressing Auxin
Signaling.,"
Science,
v.312,
2006,
p. 436.
Navarro, L; Dunoyer, P; Jay, F; Arnold, B; Dharmasiri, N; Estelle, M; Voinnet, O; Jones, JDG.
"A plant miRNA contributes to antibacterial resistance by repressing auxin signaling,"
SCIENCE,
v.312,
2006,
p. 436
- 439.
Potuschak, T; Vansiri, A; Binder, BM; Lechner, E; Vierstra, RD; Genschik, P.
"The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis,"
PLANT CELL,
v.18,
2006,
p. 3047
- 3057.
