DETERMINANTS OF RAS PROTEINS SPECIFYING THE SENSITIVITY TO YEAST IRA2P AND HUMAN P120-GAP

Human and Saccharomyces cerevisiae Ras proteins and their regulators G AP (GTPase activating protein) and GEF (guanine nucleotide exchange fa ctor) display structural similarities and are functionally interchange able in vivo and in vitro, indicating that the molecular mechanism reg ulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly s pecific for yeast Ras proteins and cannot stimulate the GTPase of mamm alian Ras. This study searches for the reasons for the different sensi tivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H- ras/Ras2p chimaeras s howed that Gly18 of Ras2p (Ala11 of H-ras p21) i s an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more cruci al determinant was found to be the 89-102 region of Ras2p (82-95 of H- ras p21) including the distal part of strand beta 4, loop L6 and the p roximal part of helix alpha 3. It was possible to construct Ras2p's re sistant to Ira2p but still sensitive to human p120-GAP and, conversely , a H-ras p21 sensitive to Ira2p. This work helps clarify specific asp ects of the conserved molecular mechanism of interaction between Ras p roteins and their negative GAP regulators.