| Attributs | Valeurs |
|---|
| type
| |
| Is Part Of
| |
| Subject
| |
| Title
| - Kinetics of Cdc42 Membrane Extraction by Rho-GDI Monitored by Real-TimeFluorescence Resonance Energy Transfer
|
| has manifestation of work
| |
| related by
| |
| Author
| |
| Abstract
| - The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) toelicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In thisreport, we have set out to address this issue by using fluorescence resonance energy transfer approachesto examine the functional interactions of the RhoGDI with membrane-associated Cdc42. Two fluorescenceassays were developed to monitor the interactions between these proteins in real time. The first involvedmeasurements of resonance energy transfer between N-methylanthraniloyl GDP (MantGDP) bound toCdc42 and fluorescein maleimide covalently attached to cysteine 79 of RhoGDI (RhoGDI-FM). Thisassay allowed us to directly monitor the binding of RhoGDI to membrane-associated Cdc42. The secondfluorescence assay involved measurements of resonance energy transfer between membrane-associatedCdc42−MantGDP and hexadecyl(amino) fluorescein that was randomly inserted into the membrane bilayer.This assay enabled us to directly monitor the (GDI-induced) release of Cdc42 from membranes. Analysesof the rates of change in the fluorescence of Cdc42−MantGDP, which serves as a resonance energytransfer donor in both of these assays, as a function of RhoGDI concentration suggests a two-step mechanismto explain the ability of RhoGDI to stimulate the release of Cdc42 from membranes. Specifically, wepropose that the GDI first binds rapidly to membrane-associated Cdc42 and then a slower isomerizationoccurs which represents the rate-limiting step for the dissociation of the Cdc42−RhoGDI complex frommembranes. We propose that this slow step in the observed kinetics reflects the time-course of translocationof the geranyl−geranyl lipid tail of Cdc42 from the outer leaflet of the membrane to the isoprenyl bindingsite observed in the previously reported NMR structure of the Cdc42−RhoGDI complex [Gosser et al.(1997) Nature 387, 814].
|
| article type
| |
| is part of this journal
| |
