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 localizat