Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae

The CDC25 gene product is a guanine nucleotide exchange factor for Ras prot eins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this h ypothesis we measured the Levels of nucleotides in yeast cells under differ ent growth conditions and the relative amount of Ras2-GTP. The intracellula r GTP/GDP ratio was found to be very sensitive to growth conditions: the ra tio is high, close to that of ATP/ABP during exponential growth, but it dec reases rapidly before the beginning of stationary phase, and it drops furth er under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of R as2-GTP changes in a parallel way suggesting that there is a correlation wi th the cytosolic GTP/GDP ratio. In addition 'in vitro' mixed-nucleotide exc hange experiments done on purified Ras2 protein demonstrated that the GTP a nd GDP concentrations influence the extent of Ras2-GTP loading giving, furt her support to their possible regulatory role. (C) 2001 Elsevier Science B. V. All rights reserved.