Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 17, Issue 6, Pages 2770-2779Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E06-01-0005
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Funding
- NCRR NIH HHS [P41 RR013186, P41 RR13186] Funding Source: Medline
- NIGMS NIH HHS [R01 GM47214, R01 GM047214, U54 GM064346, U54 GM64346] Funding Source: Medline
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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 similar to 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.
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