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Vol. 17, Issue 6, 2770-2779, June 2006
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*Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908; Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030; and Departments of Cell Biology, Microbiology, and ||Biomedical Engineering and ¶Mellon Prostate Cancer Research Center, University of Virginia, Charlottesville, VA 22908
Submitted January 3, 2006;
Revised March 14, 2006;
Accepted March 29, 2006
Monitoring Editor: Anne Ridley
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 (koff) of membrane-bound GFP-Rac. We find that koff is 0.048 s–1 for wtRac and 
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 koff 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 koff. These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Martin A. Schwartz ( maschwartz{at}virginia.edu)
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