Determinants of RasC specificity during Dictyostelium aggregation.
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, RasG(1-77)-RasC(79-189), 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))-RasC(79-189)), 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, RasC(1-78)-RasG(78-189), 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.
Bolourani
Parvin
P
Department of Microbiology and Immunology, Life Sciences Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Spiegelman
George
G
Weeks
Gerald
G
D000262:Adenylyl Cyclases / Q000378:metabolism; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D003720:Densitometry / Q000379:methods; D004023:Dictyostelium / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D015800:Protozoan Proteins; C424065:rasG protein, Dictyostelium; D000242:Cyclic AMP; D018631:ras Proteins; D000262:Adenylyl Cyclases