PROPERTIES OF THE CATALYTIC DOMAIN OF SDC25P, A YEAST GDP GTP EXCHANGE FACTOR OF RAS PROTEINS COMPLEXATION WITH WILD-TYPE RAS2P, [S24N]RAS2P AND [R80D, N81D]RAS2P

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene produc t, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-t ransferase. The highly purified (greater than 95%) stable fusion prote in, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ra s2p or human H-ras p21. This activity was further increased (three tim es) by glutathione S-transferase cleavage with thrombin. The stimulati on of the guanine nucleotide release by Sdc25p-C was stronger for Ras2 p.GDP than Ras2p.GTP, an effect that was less pronounced in the case o f the p21 complexes. The association rate of the Ras2p.GDP (GTP) compl ex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibi t the nucleotide-releasing activity of Sdc25p-C. Retention phenomena o ccurring on gel-filtration chromatography hindered the use of highly p urified Sdc25p-C to study the formation of stable complexes with Ras2p . For this purpose, Sdc25p-C was produced as a non-glutathione-S-trans ferase fusion protein via pTTQ19. Upon partial purification, this prod uct yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) s howing the same specific activity as the 64-kDa Sdc25p-C protein. On g el filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) f ormed a stable 1:1 stoichiometric complex that was dissociated by incr easing concentrations of GDP. The proper ties of this complex were ana lyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 know n to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recess ive negative mutant insensitive to the truncated form of Sdc25p-C in v itro. The complex with [S24N]Ras2p was greater than 100-fold less sens itive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These result s strongly suggest that the residues R80 and N81 are situated in or cl osely associated with the Ras2p specific site binding Sdc25p.