DIFFERENTIAL ACTIVATION OF YEAST ADENYLYL-CYCLASE BY RAS1 AND RAS2 DEPENDS ON THE CONSERVED N-TERMINUS

Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a numb er of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variab le domains in determining these differences, chimeric RAS1-RAS2 or RAS 2-RAS1 genes were constructed by swapping the sequences encoding the v ariable C-terminal domains. These constructs were expressed in a cdc25 (ts) ras1 ras2 strain, Biochemical data show that the difference ineff icacy of adenylyl cyclase activation between the two Ras proteins resi des in the highly conserved N-terminal domain. This finding is support ed by the observation that Ras2 Delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast aden ylyl cyclase than Ras1 Delta, in which the C-terminal domain of Ras1 h as been deleted. These observations suggest that amino acid residues o ther than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase, Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 Delta, Ras2, and Ras2-Ras1 strains throughou t the growth curve. This was found to result from the higher expressio n of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-term inal domains are responsible for these differences, through interactio n with other regulatory elements.