In vivo dynamics of Rac-membrane interactions.
The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.
Moissoglu
Konstadinos
K
Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.
Slepchenko
Boris M
BM
Meller
Nahum
N
Horwitz
Alan F
AF
Schwartz
Martin A
MA
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D003198:Computer Simulation; D004058:Diffusion; D017930:Genes, Reporter; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008962:Models, Theoretical; D010957:Plasmids; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D020730:Guanine Nucleotide Dissociation Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020744:rac GTP-Binding Proteins