Saccharomyces
cerevisiae
Cdc25: a still puzzling GEF.
Renata Tisi, Fiorella Belotti, Francesco Brunetti, Sonia Colombo, Enzo
Martegani
Biotecnologie e Bioscienze, Università di Milano Bicocca, P.zza della Scienza,
Milano, 20126, Italy (enzo.martegani@unimib.it)
Although Cdc25 was the first Ras-GEF (guanine nucleotide exchange factor) identified, very little is known so far about its activity regulation [1]. Recently it was suggested that the dispensable N-terminal domain could exert a negative control on the catalytic activity of the protein [2]. In order to investigate the role of this domain we constructed strains expressing only the C-terminal domain of Cdc25 (aa 876-1589), or completely lacking this protein, which was substituted with an heterologous GEF domain (Cdc25Mm or hSos1). All the strains can grow efficiently on non-fermentable carbon sources, that is in contrast with data reported in literature [3]. The deletion of the complete ORF completely abolished the carbon source dependent cell size control, in fact the cell size is the same during exponential growth on ethanol medium as in glucose. The regulation of the cell size is still present in the mutant lacking the aa 1-875 region. These results taken together suggest that the regulation of cell size on different carbon sources requires Cdc25, but the SH3 domain (aa 65-134) is not involved. Moreover, we made fusions of Cdc25 with the green fluorescent protein, and it turned out that this protein is not mainly located in the cell plasma membraner, suggesting a localisation pattern more complex than expected. [1] Rudoni S. et al. (2001). BBA 1538(2-3):181-189. [2] Chen R.A. et al. (2000). Genetics 154, 1473-1484. [3] Munder et al. (1989). FEBS Letters 242, 341-345.