Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post -translationally processed and, when co-expressed with its cytosolic inhibi tory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/R hoGDI complex, purified to greater than 98% at high yield from the yeast cy tosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridiu m botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleo tide exchanged fully with [H-3]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 x 10(-5) s(-1), considerably slower than free RhoA. Hydrol ysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyz ing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant e xpressed as a complex with RhoGDI and purified without added nucleotide als o bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Mi croinjection of the GTP-bound G14V-RhoA/RhocDI complex (but not the GDP for m) into serum-starved Swiss 3T3 cells elicited formation of stress fibers a nd focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated fro m its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These res ults show that GTP-triggered translocation of RhoA from RhoGDI to a membran e, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or mem brane receptors.