Determinants of RasC specificity during Dictyostelium aggregation

Abstract

RasC is required for optimum activation of adenylyl cyclase A and for aggregate stream formation during the early differentiation of Dictyostelium discoideum. RasG is unable to substitute for this requirement despite its sequence similarity to RasC. A critical question is which amino acids in RasC are required for its specific function. Each of the amino acids within the switch 1 and 2 domains in the N-terminal portion of RasG was changed to the corresponding amino acid from RasC, and the ability of the mutated RasG protein to reverse the phenotype of rasC- cells was determined. Only the change from aspartate at position 30 of RasG to alanine (the equivalent position 31 in RasC) resulted in a significant increase in adenylyl cyclase A activation and a partial reversal of the aggregation- deficient phenotype of rasC - cells. All other single amino acid changes were without effect. Expression of a chimeric protein, RasG1-77-RasC79-189, also resulted in a partial reversal of the rasC- cell phenotype, indicating the importance of the C-terminal portion of RasC. Furthermore, expression of the chimeric protein, with alanine changed to aspartate (RasG 1-77(D30A)-RasC79 -189), resulted in a full rescue the rasC- aggregation-deficient phenotype. Finally, the expression of either a mutated RasC, with the aspartate 31 replaced by alanine, or the chimeric protein, RasC1-78- RasG78-189, only generated a partial rescue. These results emphasize the importance of both the single amino acid at position 31 and the C-terminal sequence for the specific function of RasC during Dictyostelium aggregation. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.