Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regul atory protein involved in the cycling of Rho proteins between membrane-boun d and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free s ystem derived from rat kidney. Addition of GST-GDI to kidney brush border m embranes resulted in the specific release of Cdc42 and RhoA from the membra nes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA b y GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membran es and could not be solubilized by repeated GST-GDI treatment. These result s demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membr ane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GD I, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCI prior treatment by GST-GDI inhibited the extracti on of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological i onic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttli ng activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.