Mutational analysis of the role of Rap1 in regulating cytoskeletal function in Dictyostelium

It was shown previously that increased expression of the ras-related rap1 gene in Dictyostelium discoideum altered cell morphology (Rebstein et al., Dev. Genet 1993, 14,347-355). Vegetative Rap1 transformants were more flattened and spread than parental Ax2 cells and had increased F-actin near the cell periphery. In addition, starving Rap1 cells were inhibited in the rapid cell contraction that occurs upon refeeding with nutrient media, in this communication, we show that expression of Rap also markedly reduces the contraction response that occurs upon addition of azide to vegetative cells. The changes in cell morphology, the refeeding contraction response, and the azide contraction response have been used to analyze mutants of Rap1 generated by site-directed mutagenesis. The substitution G12V, predicted to increase the proportion of protein binding GTP, did not alter the effect of Rap on cell morphology or on its ability to inhibit the contraction response to azide, but modestly enhanced the ability of Rap1 to inhibit cell rounding in response to nutrient media, The substitution S17N, predicted to restrict the protein to the GDP-bound state, did not produce the flattened cell morphology and abolished the inhibitory effects of Rap in the two cell contraction assays. These results are consistent with a requirement of GTP binding for the Rap-induced effects. Transformants carrying the Rap-S17N protein had a more polar morphology than the parental Ax2 cells, suggesting the possibility that Rap-S17N interferes with the ability of endogenous Rap to regulate the cytoskeleton, Substitutions at amino acid 38, within the presumptive effector domain, reduced but did not abolish the effects of Rap1 on cell contraction, while the substitution T61Q had no effect on Rap1 activity. Taken together, the results suggest that Rap may have multiple regulatory effects on cytoskeletal function. (C) 1997 Academic Press.