Cdc25 protein overexpression reveals an unexpected nuclear localization.
Renata Tisi (1), Francesco Brunetti (2), Enzo Martegani (1)
(1) Dip. Biotecn. e Bioscienze, Universita' di Milano Bicocca, P.za della Scienza 2, Milan, 20126, Italy; (2) Dip. Biologia, Universita' di Milano, via Celoria 26, Milan, Italy
The mechanisms regulating the activity of Saccharomyces cerevisiae Ras-GEF Cdc25 are still largely unknown. Recently it was suggested that the dispensable N-terminal domain could exert a negative control on the protein catalytic activity (1) and that this region could be necessary for growth on non-fermentable carbon sources (2), but strains expressing only the C-terminal domain of Cdc25 (aa 876-1589), or completely lacking this protein, which was substituted by an heterologous GEF domain (Cdc25 Mm or hSos1), didn't show any growth defect (3). In order to investigate Cdc25 localization and the role of its different domains we constructed several fusions of the full length Cdc25 or of different fragments of the protein with the green fluorescent protein, and it turned out that this protein, when even slightly overexpressed, is not at all located in the cell plasma membrane. The full protein fusion, and even more the fusions spanning aa 353-875, aa 876-1100 or aa 353-1100 seem to localize mainly in the nucleus, concentrating in the nuclear periphery, in a region distinct from the nucleolus. This could be related to the presence of two nuclear localization signals (NLS) in position 547 and 806. Further insight in the meaning of this unexpected localization will be obtained by deleting the 353-1100 fragment in the full protein construct. (1) Chen et al. (2000). Genetics 154, 1473-1484. (2) Munder et al. (1989). FEBS Letters 242, 341-345. (3) Tisi et al. (2003). Yeast 20, S162.