Live-cell imaging of endogenous Ras-GTP shows predominant Ras activation at the plasma membrane and in the nucleus in Saccharomyces cerevisiae

Abstract

Ras proteins function as a point of convergence for different signalling pathways in eukaryotes and are involved in many cellular responses; their different subcellular locations could regulate distinct functions. To investigate the localization of active Ras in vivo in Saccharomyces cerevisiae, we expressed a probe consisting of a GFP fusion with a trimeric Ras binding domain of Raf1 (eGFP-RBD3), which binds Ras-GTP with a much higher affinity than Ras-GDP. Our results show that in wild type cells active Ras accumulates mainly at the plasma membrane and in the nucleus during growth on medium containing glucose, while it accumulates mainly in mitochondria in wild type glucose-starved cells and relocalizes to the plasma membrane and to the nucleus upon addition of this sugar. A similar pattern is observed in a strain deleted in the CYR1 gene indicating that the absence of adenylate cyclase does not impair the localization of Ras-GTP. Remarkably, in a gpa2Δ, but not in a gpr1Δ mutant, active Ras accumulates in internal membranes and mitochondria, both when cells are growing on glucose medium or are starved, indicating that Gpa2, but not Gpr1 is required for the recruitment of Ras-GTP at the plasma membrane and in the nucleus. Moreover, deletion of both HXK1 and HXK2 also causes a mitochondrial localization of the probe, which relocalizes to the plasma membrane and to the nucleus upon expression of HXK2 on a centromeric plasmid, suggesting that this kinase is involved in the proper localization of active Ras.