Subcellular Localization And The Mechanism Of Pde1 In The Yeast Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, whichencode a low-affinity and a high-affinity cAMP phosphodiesterase, respectively. Thelow-affinity enzyme Pde1 in yeast, has a specific function in the feedback control ofthe glucose-induced cAMP signaling upon its phosphorylation by PKA. Previousresearch has found that in vitro phosphorylation of Purified Pde1 does not change itsintrinsic activity or affinity for cAMP. Phosphorylation of Pde1 was suggested to beimportant in vivo for targeting Pde1 to a specific subcellular location where cAMP isdegraded. A PDE1-GFP fusion gene was constructed which is called V410. Thisplasmid was transformed into the yeast strains, YN123, YN100 and YN101expressing a normal, an attenuated or a constitutively higth PKA activity, respectively.The genomic copy of the PDE1 gene has been disrupted in these strains. Subcellularlocalization of the Pde1-GFP fusion protein in these strains were examined byimmunofluorescence microscopy before and after addition of glucose to cells growingexponentially on synthetic media containing nonfermentable carbon sources. Wefound that, in YN123 and YN100 cells grown on nonfermentable carbon sources,Pde1 was located in the cytoplasm, possibly bound to the endoplasmic reticulum orGolgi apparatus. In YN123 cells, addition of glucose did not cause visible change inPde1-GFP localization. In YN100 cells, however, Pde1-GFP localization werechanged from specific cytoplasmic localization (endoplasmic reticulum or Golgiapparatus) to the cytosol after addition of glucose. More interestingly, in YN101 cellsPde1-GFP signal remained constantly in the cytosol both before and within threeminutes after addition of glucose. These results implicate that in cells with low PKAactivity (cells grown on a nonfermentable carbon source or with constitutively lowPKA activity) unphosphorylated Pde1 may be sequestered from the cytosol to the ERor Golgi apparatus preventing its effect on cAMP degradation;On the other hand, incells with high PKA activity (cells growing on glucose or with constitutively highPKA activity) Pde1 is phosphorylated and targeted to the cytosol to hydrolyze cAMP.We also noticed that, like in YN123 and YN100 cells growing on nonfermentablecarbon sources, Pde1-GFP showed scattered localization in the cytoplasm 5 minutesafter addition of glucose. This is in consistence with previous results showing that,once yeast cells have started fermentative growth on glucose, the Ras-cAMP pathwaywill be turned off and another pathway called FGM pathway will take over thefunction of PKA to activate PKA targets. The Sch9 protein kinase is the only knowncomponents of the FGM pathway and has an inhibitory effect on PKA. Therefore,activation of the FGM pathway by the addition of glucose resulted in a lower PKAactivity and, as a consequence, decreased phosphorylation of Pde1. In addition, wefound that in budded cells Pde1p was more concentrated in the bud than in the mothercell and tended to accumulate at the apical growth site of the bud indicating that Pde1is preferentially localized to the most energetic parts of the cell to regulate cell growthand differentiation.