Studies on active RAS proteins localization and evidences for nuclear active RAS2 involvement in invasive growth in saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Ras proteins are part of the cAMP/PKA signalling pathway, which plays a fundamental role in the control of many cellular processes including cells proliferation, stress resistance, metabolism, and growth. They belong to the super-family of the small GTPases that act as molecular switches by cycling between an inactive GDP-bound form and an active GTP-bound form. This process is controlled by two classes of regulatory proteins: the GEFs promote the activation of Ras by catalyzing the GDP-GTP exchange, whereas the GAPs turn off the Ras proteins by stimulating the hydrolysis of GTP to GDP. In the first section of this thesis, we investigated the localization of active Ras proteins in wild type cells and in mutants in several components of the cAMP/PKA pathway to understand how the proteins involved in this pathway influence the localization of active Ras. To this aim we used a probe in which the eGFP (enhanced green fluorescent protein) is fused to a trimeric Ras binding domain (RBD3) of the human Ras effector, c-Raf1. This RBD directly binds to the active Ras with a much higher affinity than the inactive Ras. We also investigated the influence of PKA activity on active Ras localization analyzing different mutants with either high or low/absent PKA activity. The cells of the different strains expressing the eGFP-RBD3 probe growing on glucose medium were observed under the microscope. In wild type cells, Ras-GTP was mainly localized at the plasma membrane and surprisingly in the nucleus. In cyr1∆ and gpr1∆ cells, the probe showed a similar localization as in wild type cells. In gpa2∆, hxk2∆ and hxk1∆hxk2∆ cells, the fluorescence accumulated in internal membranes and mitochondria. However, in the hxk1∆hxk2∆ cells transformed with the centromeric plasmid YCpHXK2 expressing Hxk2, the eGFP-RBD3 probe was mainly localized at the plasma membrane and in the nucleus. These results suggest that Gpa2 and Hxk2 play a role in the localization of active Ras. We also observed that the localization of active Ras is dependent on PKA activity. Indeed, in the bcy1∆ mutant, showing high PKA activity, there was a clear relocalization of active Ras to the cytoplasm and to the nucleus, while no active Ras was localized at the plasma membrane anymore. In a strain with either reduced PKA activity, the tpk1w1 tpk2∆ tpk3∆ strain or absent PKA activity, the tpk1∆ tpk2∆ tpk3∆ yak1∆ strain, active Ras was mainly localized at the plasma membrane. In the second section of this thesis, we investigated the role played by active Ras in the nucleus. To this aim, a fusion was made between the Ras2 protein and the Nuclear Export Signal (NES) from the HIV virus (HIV virus Rev protein NES) (Henderson et al., 2000), generating the NES-RAS2 strain. Our results showed that the exclusion of Ras2 protein from the nucleus did not cause a growth defect neither on fermentable nor non fermentable carbon sources and did not influence the PKA related phenotypes analyzed in our work. Cells expressing the fusion protein were only defective for the invasive growth, suggesting that nuclear active Ras2 is involved in this cellular process. These results were confirmed using also the Tlys86 strain, that is commonly used to test this phenotype. We also demonstrated that the nuclear localization of Cdc25, the main GEF of Ras proteins, is required for invasive growth and that PKA activity controls invasive growth influencing the localization of active Ras. Data in literature (Cazzaniga et al., 2008; Pescini et al., 2012) show the presence in silico of cAMP levels oscillations. In the last section of this thesis, we tested two different FRET sensors, previously used in mammalian cells, to monitor the cAMP levels (CFP-Epac1-YFP probe) and PKA activity in single cells in vivo (AKAR3 probe). We inserted the sequences coding for the CFP-Epac1-YFP sensor and for the AKAR3 sensor in a multicopy yeast expression vector and the sensors were expressed under the control of the TPI promoter in several yeast strains. We used a two-photon confocal microscope system to measure the FRET efficiency. Our preliminary results showed that in a wild type strain expressing either the Epac sensor or the AKAR3 sensor there was respectively an increase of cAMP level and PKA activity in a single yeast cell after glucose addition to glucose-starved cells.