Abstract
Fractionation and extraction of nuclear proteins are techniques intended to facilitate dedicated plant proteomic studies. These techniques rely on subcellular fractionation, which makes it possible to define and characterize the proteome of a subcellular organelle, in this case the cell nucleus. Nuclear protein fractionation is proposed as a method to be carried out according to the solubility of proteins in buffers of increasing ionic strength. This physical criterion, accompanied in some steps by the use of additional reagents, such as detergents or enzymes, produces fractions that have been demonstrated to have functional significance. The proposed procedure yields five fractions, the first of them containing proteins associated with the nuclear envelope and remnants of the cytoskeleton. The second fraction, which is soluble at low ionic strength, contains ribonucleoproteins active in nuclear RNA metabolism. After increasing ionic strength and digesting with DNase, the result is the chromatin fraction. Finally, the fourth and fifth fractions correspond to the nuclear matrix and are obtained, respectively, by solubilization in high salt concentration and in the form of the residual pellet, which is only soluble in 7 M urea under sonication. This procedure offers a wide range of applicability, even in the cases in which the genome of the particular species investigated is not sequenced. In general, the functional criteria driving the extraction method described here will make this method capable of generating valuable and useful information.
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References
Gallardo, K., Job, C., Groot, S. P. C., et al. (2002) Proteomics of Arabidopsis seed germination. A comparative study of wild-type and gibberellin-deficient seeds. Plant Physiol. 129, 823–837.
Gallardo, K., Job, C., Groot, S. P. C., et al. (2001) Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol. 126, 835–848.
Prime, T. A., Sherrier, D. J., Mahon, P., Packman, L. C., and Dupree, P. (2000) A proteomic analysis of organelles from Arabidopsis thaliana. Electrophoresis 21, 3488–3499.
Shen, S., Jing, Y., and Kuang, T. (2003) Proteomics approach to identify wound-response related proteins from rice leaf sheath. Proteomics 3, 527–535.
Mathesius, U., Keijzes, G., Natera, S. H. A., Weinman, J. J., Djordevic, M. A., and Rolfe, B. G. (2001) Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting. Proteomics 1, 1424–1440.
Chivasa, S., Ndimba, B.K., Simon, W.J., et al. (2002) Proteomic analysis of the Arabidopsis thaliana cell wall. Electrophoresis 23, 1754–1765.
Shevchenko, A., Jensen, O.N., Podtelejnikov, A. V., et al. (1996) Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. USA 93, 14,440–14,445.
Busch, H., Ballal, N. R., Rao, M. R. S., Choi, Y. C., and Rothblum, L. I. (1978) Factors affecting nucleolar rDNA readouts, in The Cell Nucleus, vol. 5, Chromatin, part B. (Busch, H., ed.), Academic Press, New York, pp. 416–468.
Bourbon, H. M., Bugler, B., Caizergues-Ferrer, M., and Amalric, F. (1983) Role of phosphorylation on the maturation pathways of 100 KD nucleolar protein. FEBS Lett. 155, 218–222.
He, D., Nickerson, J. A., and Penman, S. (1990) Core filaments of the nuclear matrix. J. Cell Biol. 110, 569–580.
González-Camacho, F. and Medina, F. J. (2004) Identification of specific plant nucleolar phosphoproteins in a functional proteomic analysis. Proteomics 4, 407–417.
Matía, I., González-Camacho, F., Marco, R., Kiss, J.Z., Gasset, G., and Medina, F.J. (2005) Nucleolar structure and proliferation activity of Arabidopsis root cells from seedlings germinated on the International Space Station. Adv. Space Res. 36, 1244–1253.
Greimers, R. and Deltour, R. (1981) Organization of transcribed and nontranscribed chromatin in isolated nuclei of Zea mays root cells. Eur. J. Cell Biol. 23, 303–311.
De Cárcer, G., Cerdido, A., and Medina, F. J. (1997) NopA64, a novel nucleolar phosphoprotein from proliferating onion cells, sharing immunological determinants with mammalian nucleolin. Planta 201, 487–495.
De Cárcer, G., Lallena, M. J., and Correas, I. (1995) Protein 4.1 is a component of the nuclear matrix of mammalian cells. Biochem. J. 312, 871–877.
Schnapp, A., Pfliderer, C., Rosenbauer, H., and Grummt, I. (1990) A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis. EMBO J. 9, 2857–2863.
Dunham, V. L. and Bryant, J. A. (1983) Nuclei, in Isolation of Membranes and Organelles from Plant Cells (Hall, J. L. and Moore, L., eds.), Academic Press, London, pp. 237–275.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 7, 248–254.
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González-Camacho, F., Medina, F.J. (2007). Extraction of Nuclear Proteins from Root Meristematic Cells. In: Thiellement, H., Zivy, M., Damerval, C., Méchin, V. (eds) Plant Proteomics. Methods in Molecular Biology, vol 355. Humana Press. https://doi.org/10.1385/1-59745-227-0:63
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DOI: https://doi.org/10.1385/1-59745-227-0:63
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