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 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.
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Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22906-0011, USA.
Liu
X
X
Longenecker
K
K
Dipierro
C G
CG
Walker
L A
LA
Somlyo
A V
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Somlyo
A P
AP
Nakamoto
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RK
D016475:3T3 Cells; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D051379:Mice; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C569638:ARHGDIA protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D008081:Liposomes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020742:rhoA GTP-Binding Protein