Wilhelm bernhard workshop on the cell nucleus

FUNCTIONAL PROTEOMIC ANALYSIS OF PLANT NUCLEOLAR PHOSPHOPROTEINS RELATED TO CELL PROLIFERATION

González-Camacho, F., and Medina, F.J.

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in the nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing 2D- proteomes obtained from the soluble fraction of onion nuclei in actively proliferating root meristematic cells versus non-meristematic root cells. In particular, we have studied the physico-chemical features of the major nucleolar protein NopA100, a highly phosphorylated nucleolin-like protein. A total of 388 spots were quantified in meristematic nuclei, while only 215 were detected in non-meristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, relevant well-known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin-like protein NopA100, exhibited a significant increase in proliferating cells. By Western blotting with anti-NopA100 antibody, 17 spots, 100 kDa in molecular mass, were detected in the meristematic sample. All the spots detected form a cluster through a pI range of 4.3-6.6. This cluster gives account of different states of phosphorylation exhibited by the protein in dependence of the nucleolar activity and the cell cycle phases. On the contrary, only 8 spots were found in the extract from non-meristematic nuclei, whose pI range was shortened to 4.8-6.1. This indicates a substantially lower amount of phosphorylation variants, associated with the drop of the proliferation capacity and of the nucleolar activity. We have also analyzed the bidimensional AgNOR staining pattern of this fraction in order to identify in plant cells the stained protein spots, which are commonly recognized to be phosphoproteins, markers of proliferation. Other than corroborating that NopA100, the onion nucleolin-like protein, is an Ag-NOR protein, we were interested in the characterization of the plant homologue to the nucleolar phosphoprotein B23. It was revealed as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. These features are not totally coincident with those described for mammalian cells. The use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are an advantageous alternative to random selection procedures for proteomic studies involving further mass spectrometry analysis.