Methods to study the Ras2 protein activation state and the subcellular localization of Ras-GTP in Saccharomyces cerevisiae.
Ras proteins were highly conserved during evolution. They function as a point of convergence for different signalling pathways in eukaryotes and are involved in a wide range of cellular responses (shift from gluconeogenic to fermentative growth, breakdown of storage carbohydrates, stress resistance, growth control and determination of life span, morphogenesis and development, and others). These proteins are members of the small GTPase superfamily, which are active in the GTP-bound form and inactive in the GDP-bound form. Given the importance of studies on the Ras protein activation state to understand the detailed mechanism of Ras-mediated signal transduction, we provide here a simple, sensitive, and reliable method, based on the high affinity interaction of Ras-GTP with the Ras binding domain (RBD) of Raf1, to measure the level of Ras2-GTP on total Ras2 in Saccharomyces cerevisiae. Moreover, to study the localization of Ras-GTP in vivo in single S. cerevisiae cells, we expressed a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3), which was proven to be a useful live-cell biosensor for Ras-GTP in mammalian cells.
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Sonia
S
Colombo
Enzo
E
Martegani
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014170:Transformation, Genetic; D014617:Vacuoles / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D017382:Reactive Oxygen Species; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins