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

Protein Sci. 2000 Feb;9(2):376-86. doi: 10.1110/ps.9.2.376.


The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]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. 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-RhoA/RhoGDI 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/RhoGDI complex that does not require other protein factors or membrane receptors.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adenosine Diphosphate Ribose / metabolism
  • Animals
  • Biological Transport, Active
  • Guanine Nucleotide Dissociation Inhibitors / chemistry
  • Guanine Nucleotide Dissociation Inhibitors / genetics
  • Guanine Nucleotide Dissociation Inhibitors / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / metabolism*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Liposomes
  • Macromolecular Substances
  • Mice
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Signal Transduction
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors
  • rhoA GTP-Binding Protein / chemistry
  • rhoA GTP-Binding Protein / genetics
  • rhoA GTP-Binding Protein / metabolism*


  • ARHGDIA protein, human
  • Guanine Nucleotide Dissociation Inhibitors
  • Liposomes
  • Macromolecular Substances
  • Recombinant Proteins
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors
  • Guanosine Diphosphate
  • Adenosine Diphosphate Ribose
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate
  • rhoA GTP-Binding Protein