Evidence for a role for the Dictyostelium Rap1 in cell viability and the response to osmotic stress

Abstract

The Dictyostelium genome contains a single rapA gene,which encodes a Rap1 monomeric G protein. As attempts at generating rapA-null Dictyostelium cells had been unsuccessful,expression of antisense RNA from the rapA gene under control of the folate repressible discoidin promoter was used to reduce cellular levels of the Rap1 protein. As Rap1 levels gradually decreased following antisense rapA RNA induction, growth rate and cell viability also decreased, a result consistent with the idea that rapA is an essential gene. The Rap1-depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4 M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a role for Rap1 in the generation of cGMP. Dictyostelium Rap1 formed a complex with the Ras-binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4 M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2 mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature but not in response to low osmolarity or high temperature.