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 cerevisiaeis post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA0 RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinumC3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with @ 3 H#GDP or partially with GTP in the presence of submicromolar Mg 21 . The GTP-RhoA0 RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 3 10 25 s 21 , considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA0RhoGDI complex ~but not the GDP form! into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA 0RhoGDI complex that does not require other protein factors or membrane receptors.